Release minimap2-2.13 (r850)

I don't know why I haven't found it at the beginning.

.gitignore

@@ -1,4 +1,8 @@
+.cproject
+.project
 .*.swp
 *.a
 *.o
 *.dSYM
+minimap2
+mappy.c

.travis.yml

@@ -1,5 +1,20 @@
-language: c
-compiler:
-  - gcc
-  - clang
-script: make
+matrix:
+  include:
+    - language: c
+      compiler: gcc
+      script: make
+    - language: c
+      compiler: clang
+      script: make
+    - language: python
+      python: "2.7"
+      before_install: pip install cython
+      script: python setup.py build_ext
+    - language: python
+      python: "3.5"
+      before_install: pip install cython
+      script: python setup.py build_ext
+    - language: python
+      python: "3.6"
+      before_install: pip install cython
+      script: python setup.py build_ext

MANIFEST.in

@@ -0,0 +1,11 @@
+include *.h
+include Makefile
+include ksw2_dispatch.c
+include main.c
+include README.md
+include sse2neon/emmintrin.h
+include python/mappy.c
+include python/cmappy.h
+include python/cmappy.pxd
+include python/mappy.pyx
+include python/README.rst

Makefile

@@ -1,17 +1,28 @@
-CC=			gcc
-CFLAGS=		-g -Wall -O2 -Wc++-compat
+CFLAGS=		-g -Wall -O2 -Wc++-compat #-Wextra
 CPPFLAGS=	-DHAVE_KALLOC
-INCLUDES=	-I.
-OBJS=		kthread.o kalloc.o ksw2_extz2_sse.o ksw2_extd2_sse.o ksw2_exts2_sse.o ksw2_ll_sse.o \
-			misc.o bseq.o sketch.o sdust.o index.o chain.o align.o hit.o map.o format.o
+INCLUDES=
+OBJS=		kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o chain.o align.o hit.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o
 PROG=		minimap2
 PROG_EXTRA=	sdust minimap2-lite
 LIBS=		-lm -lz -lpthread
 
-ifeq ($(sse2only),)
-	CFLAGS+=-msse4
+ifeq ($(arm_neon),) # if arm_neon is not defined
+ifeq ($(sse2only),) # if sse2only is not defined
+	OBJS+=ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
+else                # if sse2only is defined
+	OBJS+=ksw2_extz2_sse.o ksw2_extd2_sse.o ksw2_exts2_sse.o
+endif
+else				# if arm_neon is defined
+	OBJS+=ksw2_extz2_neon.o ksw2_extd2_neon.o ksw2_exts2_neon.o
+    INCLUDES+=-Isse2neon
+ifeq ($(aarch64),)	#if aarch64 is not defined
+	CFLAGS+=-D_FILE_OFFSET_BITS=64 -mfpu=neon -fsigned-char
+else				#if aarch64 is defined
+	CFLAGS+=-D_FILE_OFFSET_BITS=64 -fsigned-char
+endif
 endif
 
+.PHONY:all extra clean depend
 .SUFFIXES:.c .o
 
 .c.o:
@@ -22,7 +33,7 @@ all:$(PROG)
 extra:all $(PROG_EXTRA)
 
 minimap2:main.o libminimap2.a
-		$(CC) $(CFLAGS) $< -o $@ -L. -lminimap2 $(LIBS)
+		$(CC) $(CFLAGS) main.o -o $@ -L. -lminimap2 $(LIBS)
 
 minimap2-lite:example.o libminimap2.a
 		$(CC) $(CFLAGS) $< -o $@ -L. -lminimap2 $(LIBS)
@@ -30,11 +41,52 @@ minimap2-lite:example.o libminimap2.a
 libminimap2.a:$(OBJS)
 		$(AR) -csru $@ $(OBJS)
 
-sdust:sdust.c kalloc.o kalloc.h kdq.h kvec.h kseq.h sdust.h
+sdust:sdust.c kalloc.o kalloc.h kdq.h kvec.h kseq.h ketopt.h sdust.h
 		$(CC) -D_SDUST_MAIN $(CFLAGS) $< kalloc.o -o $@ -lz
 
+# SSE-specific targets on x86/x86_64
+
+ifeq ($(arm_neon),)   # if arm_neon is defined, compile this target with the default setting (i.e. no -msse2)
+ksw2_ll_sse.o:ksw2_ll_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) -msse2 $(CPPFLAGS) $(INCLUDES) $< -o $@
+endif
+
+ksw2_extz2_sse41.o:ksw2_extz2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) -msse4.1 $(CPPFLAGS) -DKSW_CPU_DISPATCH $(INCLUDES) $< -o $@
+
+ksw2_extz2_sse2.o:ksw2_extz2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) -msse2 -mno-sse4.1 $(CPPFLAGS) -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY $(INCLUDES) $< -o $@
+
+ksw2_extd2_sse41.o:ksw2_extd2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) -msse4.1 $(CPPFLAGS) -DKSW_CPU_DISPATCH $(INCLUDES) $< -o $@
+
+ksw2_extd2_sse2.o:ksw2_extd2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) -msse2 -mno-sse4.1 $(CPPFLAGS) -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY $(INCLUDES) $< -o $@
+
+ksw2_exts2_sse41.o:ksw2_exts2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) -msse4.1 $(CPPFLAGS) -DKSW_CPU_DISPATCH $(INCLUDES) $< -o $@
+
+ksw2_exts2_sse2.o:ksw2_exts2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) -msse2 -mno-sse4.1 $(CPPFLAGS) -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY $(INCLUDES) $< -o $@
+
+ksw2_dispatch.o:ksw2_dispatch.c ksw2.h
+		$(CC) -c $(CFLAGS) -msse4.1 $(CPPFLAGS) -DKSW_CPU_DISPATCH $(INCLUDES) $< -o $@
+
+# NEON-specific targets on ARM
+
+ksw2_extz2_neon.o:ksw2_extz2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) $(CPPFLAGS) -DKSW_SSE2_ONLY -D__SSE2__ $(INCLUDES) $< -o $@
+
+ksw2_extd2_neon.o:ksw2_extd2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) $(CPPFLAGS) -DKSW_SSE2_ONLY -D__SSE2__ $(INCLUDES) $< -o $@
+
+ksw2_exts2_neon.o:ksw2_exts2_sse.c ksw2.h kalloc.h
+		$(CC) -c $(CFLAGS) $(CPPFLAGS) -DKSW_SSE2_ONLY -D__SSE2__ $(INCLUDES) $< -o $@
+
+# other non-file targets
+
 clean:
-		rm -fr gmon.out *.o a.out $(PROG) $(PROG_EXTRA) *~ *.a *.dSYM session*
+		rm -fr gmon.out *.o a.out $(PROG) $(PROG_EXTRA) *~ *.a *.dSYM build dist mappy*.so mappy.c python/mappy.c mappy.egg*
 
 depend:
 		(LC_ALL=C; export LC_ALL; makedepend -Y -- $(CFLAGS) $(CPPFLAGS) -- *.c)
@@ -42,19 +94,25 @@ depend:
 # DO NOT DELETE
 
 align.o: minimap.h mmpriv.h bseq.h ksw2.h kalloc.h
-bseq.o: bseq.h kseq.h
+bseq.o: bseq.h kvec.h kalloc.h kseq.h
 chain.o: minimap.h mmpriv.h bseq.h kalloc.h
+esterr.o: mmpriv.h minimap.h bseq.h
 example.o: minimap.h kseq.h
 format.o: kalloc.h mmpriv.h minimap.h bseq.h
-hit.o: mmpriv.h minimap.h bseq.h kalloc.h
+hit.o: mmpriv.h minimap.h bseq.h kalloc.h khash.h
 index.o: kthread.h bseq.h minimap.h mmpriv.h kvec.h kalloc.h khash.h
 kalloc.o: kalloc.h
 ksw2_extd2_sse.o: ksw2.h kalloc.h
 ksw2_exts2_sse.o: ksw2.h kalloc.h
 ksw2_extz2_sse.o: ksw2.h kalloc.h
 ksw2_ll_sse.o: ksw2.h kalloc.h
-main.o: bseq.h minimap.h mmpriv.h
-map.o: kthread.h kvec.h kalloc.h sdust.h mmpriv.h minimap.h bseq.h
-misc.o: minimap.h ksort.h
-sdust.o: kalloc.h kdq.h kvec.h sdust.h
-sketch.o: kvec.h kalloc.h minimap.h
+kthread.o: kthread.h
+main.o: bseq.h minimap.h mmpriv.h ketopt.h
+map.o: kthread.h kvec.h kalloc.h sdust.h mmpriv.h minimap.h bseq.h khash.h
+map.o: ksort.h
+misc.o: mmpriv.h minimap.h bseq.h ksort.h
+options.o: mmpriv.h minimap.h bseq.h
+pe.o: mmpriv.h minimap.h bseq.h kvec.h kalloc.h ksort.h
+sdust.o: kalloc.h kdq.h kvec.h ketopt.h sdust.h
+sketch.o: kvec.h kalloc.h mmpriv.h minimap.h bseq.h
+splitidx.o: mmpriv.h minimap.h bseq.h

NEWS.md

@@ -1,3 +1,408 @@
+Release 2.13-r850 (11 October 2018)
+-----------------------------------
+
+Changes to minimap2:
+
+ * Fixed wrongly formatted SAM when -L is in use (#231 and #233).
+
+ * Fixed an integer overflow in rare cases.
+
+ * Added --hard-mask-level to fine control split alignments (#244).
+
+ * Made --MD work with spliced alignment (#139).
+
+ * Replaced musl's getopt with ketopt for portability.
+
+ * Log peak memory usage on exit.
+
+This release should produce alignments identical to v2.12 and v2.11.
+
+(2.13: 11 October 2018, r850)
+
+
+
+Release 2.12-r827 (6 August 2018)
+---------------------------------
+
+Changes to minimap2:
+
+ * Added option --split-prefix to write proper alignments (correct mapping
+   quality and clustered query sequences) given a multi-part index (#141 and
+   #189; mostly by @hasindu2008).
+
+ * Fixed a memory leak when option -y is in use.
+
+Changes to mappy:
+
+ * Support the MD/cs tag (#183 and #203).
+
+ * Allow mappy to index a single sequence, to add extra flags and to change the
+   scoring system.
+
+Minimap2 should produce alignments identical to v2.11.
+
+(2.12: 6 August 2018, r827)
+
+
+
+Release 2.11-r797 (20 June 2018)
+--------------------------------
+
+Changes to minimap2:
+
+ * Improved alignment accuracy in low-complexity regions for SV calling. Thank
+   @armintoepfer for multiple offline examples.
+
+ * Added option --eqx to encode sequence match/mismatch with the =/X CIGAR
+   operators (#156, #157 and #175).
+
+ * When compiled with VC++, minimap2 generated wrong alignments due to a
+   comparison between a signed integer and an unsigned integer (#184). Also
+   fixed warnings reported by "clang -Wextra".
+
+ * Fixed incorrect anchor filtering due to a missing 64- to 32-bit cast.
+
+ * Fixed incorrect mapping quality for inversions (#148).
+
+ * Fixed incorrect alignment involving ambiguous bases (#155).
+
+ * Fixed incorrect presets: option `-r 2000` is intended to be used with
+   ava-ont, not ava-pb. The bug was introduced in 2.10.
+
+ * Fixed a bug when --for-only/--rev-only is used together with --sr or
+   --heap-sort=yes (#166).
+
+ * Fixed option -Y that was not working in the previous releases.
+
+ * Added option --lj-min-ratio to fine control the alignment of long gaps
+   found by the "long-join" heuristic (#128).
+
+ * Exposed `mm_idx_is_idx`, `mm_idx_load` and `mm_idx_dump` C APIs (#177).
+   Also fixed a bug when indexing without reference names (this feature is not
+   exposed to the command line).
+
+Changes to mappy:
+
+ * Added `__version__` (#165).
+
+ * Exposed the maximum fragment length parameter to mappy (#174).
+
+Changes to paftools:
+
+ * Don't crash when there is no "cg" tag (#153).
+
+ * Fixed wrong coverage report by "paftools.js call" (#145).
+
+This version may produce slightly different base-level alignment. The overall
+alignment statistics should remain similar.
+
+(2.11: 20 June 2018, r797)
+
+
+
+Release 2.10-r761 (27 March 2018)
+---------------------------------
+
+Changes to minimap2:
+
+ * Optionally output the MD tag for compatibility with existing tools (#63,
+   #118 and #137).
+
+ * Use SSE compiler flags more precisely to prevent compiling errors on certain
+   machines (#127).
+
+ * Added option --min-occ-floor to set a minimum occurrence threshold. Presets
+   intended for assembly-to-reference alignment set this option to 100. This
+   option alleviates issues with regions having high copy numbers (#107).
+
+ * Exit with non-zero code on file writing errors (e.g. disk full; #103 and
+   #132).
+
+ * Added option -y to copy FASTA/FASTQ comments in query sequences to the
+   output (#136).
+
+ * Added the asm20 preset for alignments between genomes at 5-10% sequence
+   divergence.
+
+ * Changed the band-width in the ava-ont preset from 500 to 2000. Oxford
+   Nanopore reads may contain long deletion sequencing errors that break
+   chaining.
+
+Changes to mappy, the Python binding:
+
+ * Fixed a typo in Align.seq() (#126).
+
+Changes to paftools.js, the companion script:
+
+ * Command sam2paf now converts the MD tag to cs.
+
+ * Support VCF output for assembly-to-reference variant calling (#109).
+
+This version should produce identical alignment for read overlapping, RNA-seq
+read mapping, and genomic read mapping. We have also added a cook book to show
+the variety uses of minimap2 on real datasets. Please see cookbook.md in the
+minimap2 source code directory.
+
+(2.10: 27 March 2017, r761)
+
+
+
+Release 2.9-r720 (23 February 2018)
+-----------------------------------
+
+This release fixed multiple minor bugs.
+
+* Fixed two bugs that lead to incorrect inversion alignment. Also improved the
+  sensitivity to small inversions by using double Z-drop cutoff (#112).
+
+* Fixed an issue that may cause the end of a query sequence unmapped (#104).
+
+* Added a mappy API to retrieve sequences from the index (#126) and to reverse
+  complement DNA sequences. Fixed a bug where the `best_n` parameter did not
+  work (#117).
+
+* Avoided segmentation fault given incorrect FASTQ input (#111).
+
+* Combined all auxiliary javascripts to paftools.js. Fixed several bugs in
+  these scripts at the same time.
+
+(2.9: 24 February 2018, r720)
+
+
+
+Release 2.8-r672 (1 February 2018)
+----------------------------------
+
+Notable changes in this release include:
+
+ * Speed up short-read alignment by ~10%. The overall mapping accuracy stays
+   the same, but the output alignments are not always identical to v2.7 due to
+   unstable sorting employed during chaining. Long-read alignment is not
+   affected by this change as the speedup is short-read specific.
+
+ * Mappy now supports paired-end short-read alignment (#87). Please see
+   python/README.rst for details.
+
+ * Added option --for-only and --rev-only to perform alignment against the
+   forward or the reverse strand of the reference genome only (#91).
+
+ * Alleviated the issue with undesired diagonal alignment in the self mapping
+   mode (#10). Even if the output is not ideal, it should not interfere with
+   other alignments. Fully resolving the issue is intricate and may require
+   additional heuristic thresholds.
+
+ * Enhanced error checking against incorrect input (#92 and #96).
+
+For long query sequences, minimap2 should output identical alignments to v2.7.
+
+(2.8: 1 February 2018, r672)
+
+
+
+Release 2.7-r654 (9 January 2018)
+---------------------------------
+
+This release fixed a bug in the splice mode and added a few minor features:
+
+ * Fixed a bug that occasionally takes an intron as a long deletion in the
+   splice mode. This was caused by wrong backtracking at the last CIGAR
+   operator. The current fix eliminates the error, but it is not optimal in
+   that it often produces a wrong junction when the last operator is an intron.
+   A future version of minimap2 may improve upon this.
+
+ * Support high-end ARM CPUs that implement the NEON instruction set (#81).
+   This enables minimap2 to work on Raspberry Pi 3 and Odroid XU4.
+
+ * Added a C API to construct a minimizer index from a set of C strings (#80).
+
+ * Check scoring specified on the command line (#79). Due to the 8-bit limit,
+   excessively large score penalties fail minimap2.
+
+For genomic sequences, minimap2 should give identical alignments to v2.6.
+
+(2.7: 9 January 2018, r654)
+
+
+
+Release 2.6-r623 (12 December 2017)
+-----------------------------------
+
+This release adds several features and fixes two minor bugs:
+
+ * Optionally build an index without sequences. This helps to reduce the
+   peak memory for read overlapping and is automatically applied when
+   base-level alignment is not requested.
+
+ * Approximately estimate per-base sequence divergence (i.e. 1-identity)
+   without performing base-level alignment, using a MashMap-like method. The
+   estimate is written to a new dv:f tag.
+
+ * Reduced the number of tiny terminal exons in RNA-seq alignment. The current
+   setting is conservative. Increase --end-seed-pen to drop more such exons.
+
+ * Reduced the peak memory when aligning long query sequences.
+
+ * Fixed a bug that is caused by HPC minimizers longer than 256bp. This should
+   have no effect in practice, but it is recommended to rebuild HPC indices if
+   possible.
+
+ * Fixed a bug when identifying identical hits (#71). This should only affect
+   artifactual reference consisting of near identical sequences.
+
+For genomic sequences, minimap2 should give nearly identical alignments to
+v2.5, except the new dv:f tag.
+
+(2.6: 12 December 2017, r623)
+
+
+
+Release 2.5-r572 (11 November 2017)
+-----------------------------------
+
+This release fixes several bugs and brings a couple of minor improvements:
+
+ * Fixed a severe bug that leads to incorrect mapping coordinates in rare
+   corner cases.
+
+ * Fixed underestimated mapping quality for chimeric alignments when the whole
+   query sequence contain many repetitive minimizers, and for chimeric
+   alignments caused by Z-drop.
+
+ * Fixed two bugs in Python binding: incorrect strand field (#57) and incorrect
+   sequence names for Python3 (#55).
+
+ * Improved mapping accuracy for highly overlapping paired ends.
+
+ * Added option -Y to use soft clipping for supplementary alignments (#56).
+
+(2.5: 11 November 2017, r572)
+
+
+
+Release 2.4-r555 (6 November 2017)
+----------------------------------
+
+As is planned, this release focuses on fine tuning the base algorithm. Notable
+changes include
+
+ * Changed the mapping quality scale to match the scale of BWA-MEM. This makes
+   minimap2 and BWA-MEM achieve similar sensitivity-specificity balance on real
+   short-read data.
+
+ * Improved the accuracy of splice alignment by modeling one additional base
+   close to the GT-AG signal. This model is used by default with `-x splice`.
+   For SIRV control data, however, it is recommended to add `--splice-flank=no`
+   to disable this feature as the SIRV splice signals are slightly different.
+
+ * Tuned the parameters for Nanopore Direct RNA reads. The recommended command
+   line is `-axsplice -k14 -uf` (#46).
+
+ * Fixed a segmentation fault when aligning PacBio reads (#47 and #48). This
+   bug is very rare but it affects all versions of minimap2. It is also
+   recommended to re-index reference genomes created with `map-pb`. For human,
+   two minimizers in an old index are wrong.
+
+ * Changed option `-L` in sync with the final decision of hts-specs: a fake
+   CIGAR takes the form of `<readLen>S<refLen>N`. Note that `-L` only enables
+   future tools to recognize long CIGARs. It is not possible for older tools to
+   work with such alignments in BAM (#43 and #51).
+
+ * Fixed a tiny issue whereby minimap2 may waste 8 bytes per candidate
+   alignment.
+
+The minimap2 technical note hosted at arXiv has also been updated to reflect
+recent changes.
+
+(2.4: 6 November 2017, r555)
+
+
+
+Release 2.3-r531 (22 October 2017)
+----------------------------------
+
+This release come with many improvements and bug fixes:
+
+ * The **sr** preset now supports paired-end short-read alignment. Minimap2 is
+   3-4 times as fast as BWA-MEM, but is slightly less accurate on simulated
+   reads.
+
+ * Meticulous improvements to assembly-to-assembly alignment (special thanks to
+   Alexey Gurevich from the QUAST team): a) apply a small penalty to matches
+   between ambiguous bases; b) reduce missing alignments due to spurious
+   overlaps; c) introduce the short form of the `cs` tag, an improvement to the
+   SAM MD tag.
+
+ * Make sure gaps are always left-aligned.
+
+ * Recognize `U` bases from Oxford Nanopore Direct RNA-seq (#33).
+
+ * Fixed slightly wrong chaining score. Fixed slightly inaccurate coordinates
+   for split alignment.
+
+ * Fixed multiple reported bugs: 1) wrong reference name for inversion
+   alignment (#30); 2) redundant SQ lines when multiple query files are
+   specified (#39); 3) non-functioning option `-K` (#36).
+
+This release has implemented all the major features I planned five months ago,
+with the addition of spliced long-read alignment. The next couple of releases
+will focus on fine tuning of the base algorithms.
+
+(2.3: 22 October 2017, r531)
+
+
+
+Release 2.2-r409 (17 September 2017)
+------------------------------------
+
+This is a feature release. It improves single-end short-read alignment and
+comes with Python bindings. Detailed changes include:
+
+ * Added the **sr** preset for single-end short-read alignment. In this mode,
+   minimap2 runs faster than BWA-MEM, but is slightly less accurate on
+   simulated data sets. Paired-end alignment is not supported as of now.
+
+ * Improved mapping quality estimate with more accurate identification of
+   repetitive hits. This mainly helps short-read alignment.
+
+ * Implemented **mappy**, a Python binding for minimap2, which is available
+   from PyPI and can be installed with `pip install --user mappy`. Python users
+   can perform read alignment without the minimap2 executable.
+
+ * Restructured the indexing APIs and documented key minimap2 APIs in the
+   header file minimap.h. Updated example.c with the new APIs. Old APIs still
+   work but may become deprecated in future.
+
+This release may output alignments different from the previous version, though
+the overall alignment statistics, such as the number of aligned bases and long
+gaps, remain close.
+
+(2.2: 17 September 2017, r409)
+
+
+
+Release 2.1.1-r341 (6 September 2017)
+-------------------------------------
+
+This is a maintenance release that is expected to output identical alignment to
+v2.1. Detailed changes include:
+
+ * Support CPU dispatch. By default, minimap2 is compiled with both SSE2 and
+   SSE4 based implementation of alignment and automatically chooses the right
+   one at runtime. This avoids unexpected errors on older CPUs (#21).
+
+ * Improved Windows support as is requested by Oxford Nanopore (#19). Minimap2
+   now avoids variable-length stacked arrays, eliminates alloca(), ships with
+   getopt_long() and provides timing functions implemented with Windows APIs.
+
+ * Fixed a potential segmentation fault when specifying -k/-w/-H with
+   multi-part index (#23).
+
+ * Fixed two memory leaks in example.c
+
+(2.1.1: 6 September 2017, r341)
+
+
+
 Release 2.1-r311 (25 August 2017)
 ---------------------------------
 

README.md

@@ -1,54 +1,277 @@
+[![GitHub Downloads](https://img.shields.io/github/downloads/lh3/minimap2/total.svg?style=social&logo=github&label=Download)](https://github.com/lh3/minimap2/releases)
+[![BioConda Install](https://img.shields.io/conda/dn/bioconda/minimap2.svg?style=flag&label=BioConda%20install)](https://anaconda.org/bioconda/minimap2)
+[![PyPI](https://img.shields.io/pypi/v/mappy.svg?style=flat)](https://pypi.python.org/pypi/mappy)
 [![Build Status](https://travis-ci.org/lh3/minimap2.svg?branch=master)](https://travis-ci.org/lh3/minimap2)
-## Getting Started
+## <a name="started"></a>Getting Started
 ```sh
 git clone https://github.com/lh3/minimap2
 cd minimap2 && make
-# long reads against a reference genome
-./minimap2 -ax map10k test/MT-human.fa test/MT-orang.fa > test.sam
+# long sequences against a reference genome
+./minimap2 -a test/MT-human.fa test/MT-orang.fa > test.sam
 # create an index first and then map
-./minimap2 -x map10k -d MT-human.mmi test/MT-human.fa
-./minimap2 -ax map10k MT-human.mmi test/MT-orang.fa > test.sam
-# long-read overlap (no test data)
-./minimap2 -x ava-pb your-reads.fa your-reads.fa > overlaps.paf
-# man page
+./minimap2 -d MT-human.mmi test/MT-human.fa
+./minimap2 -a MT-human.mmi test/MT-orang.fa > test.sam
+# use presets (no test data)
+./minimap2 -ax map-pb ref.fa pacbio.fq.gz > aln.sam       # PacBio genomic reads
+./minimap2 -ax map-ont ref.fa ont.fq.gz > aln.sam         # Oxford Nanopore genomic reads
+./minimap2 -ax asm20 ref.fa pacbio-ccs.fq.gz > aln.sam    # PacBio CCS genomic reads
+./minimap2 -ax sr ref.fa read1.fa read2.fa > aln.sam      # short genomic paired-end reads
+./minimap2 -ax splice ref.fa rna-reads.fa > aln.sam       # spliced long reads (strand unknown)
+./minimap2 -ax splice -uf -k14 ref.fa reads.fa > aln.sam  # noisy Nanopore Direct RNA-seq
+./minimap2 -ax splice -uf -C5 ref.fa query.fa > aln.sam   # Final PacBio Iso-seq or traditional cDNA
+./minimap2 -cx asm5 asm1.fa asm2.fa > aln.paf             # intra-species asm-to-asm alignment
+./minimap2 -x ava-pb reads.fa reads.fa > overlaps.paf     # PacBio read overlap
+./minimap2 -x ava-ont reads.fa reads.fa > overlaps.paf    # Nanopore read overlap
+# man page for detailed command line options
 man ./minimap2.1
 ```
+## Table of Contents
 
-## Introduction
+- [Getting Started](#started)
+- [Users' Guide](#uguide)
+  - [Installation](#install)
+  - [General usage](#general)
+  - [Use cases](#cases)
+    - [Map long noisy genomic reads](#map-long-genomic)
+    - [Map long mRNA/cDNA reads](#map-long-splice)
+    - [Find overlaps between long reads](#long-overlap)
+    - [Map short accurate genomic reads](#short-genomic)
+    - [Full genome/assembly alignment](#full-genome)
+  - [Advanced features](#advanced)
+    - [Working with >65535 CIGAR operations](#long-cigar)
+    - [The cs optional tag](#cs)
+    - [Working with the PAF format](#paftools)
+  - [Algorithm overview](#algo)
+  - [Getting help](#help)
+  - [Citing minimap2](#cite)
+- [Developers' Guide](#dguide)
+- [Limitations](#limit)
 
-Minimap2 is a fast sequence mapping and alignment program that can find
-overlaps between long noisy reads, or map long reads or their assemblies to a
-reference genome optionally with detailed alignment (i.e. CIGAR). At present,
-it works efficiently with query sequences from a few kilobases to ~100
-megabases in length at an error rate ~15%. Minimap2 outputs in the [PAF][paf] or
-the [SAM format][sam]. On limited test data sets, minimap2 is over 20 times
-faster than most other long-read aligners. It will replace BWA-MEM for long
-reads and contig alignment.
+## <a name="uguide"></a>Users' Guide
 
-Minimap2 is the successor of [minimap][minimap]. It uses a similar
-minimizer-based indexing and seeding algorithm, and improves the original
-minimap with homopolyer-compressed k-mers (see also [SMARTdenovo][smartdenovo]
-and [longISLND][longislnd]), better chaining and the ability to produce CIGAR
-with fast extension alignment (see also [libgaba][gaba] and [ksw2][ksw2]) and
-piece-wise affine gap cost.
+Minimap2 is a versatile sequence alignment program that aligns DNA or mRNA
+sequences against a large reference database. Typical use cases include: (1)
+mapping PacBio or Oxford Nanopore genomic reads to the human genome; (2)
+finding overlaps between long reads with error rate up to ~15%; (3)
+splice-aware alignment of PacBio Iso-Seq or Nanopore cDNA or Direct RNA reads
+against a reference genome; (4) aligning Illumina single- or paired-end reads;
+(5) assembly-to-assembly alignment; (6) full-genome alignment between two
+closely related species with divergence below ~15%.
 
-If you use minimap2 in your work, please consider to cite:
+For ~10kb noisy reads sequences, minimap2 is tens of times faster than
+mainstream long-read mappers such as BLASR, BWA-MEM, NGMLR and GMAP. It is more
+accurate on simulated long reads and produces biologically meaningful alignment
+ready for downstream analyses. For >100bp Illumina short reads, minimap2 is
+three times as fast as BWA-MEM and Bowtie2, and as accurate on simulated data.
+Detailed evaluations are available from the [minimap2 paper][doi] or the
+[preprint][preprint].
 
-> Li, H. (2017). Minimap2: fast pairwise alignment for long DNA sequences. [arXiv:1708.01492](https://arxiv.org/abs/1708.01492).
+### <a name="install"></a>Installation
 
-## Installation
+Minimap2 is optimized for x86-64 CPUs. You can acquire precompiled binaries from
+the [release page][release] with:
+```sh
+curl -L https://github.com/lh3/minimap2/releases/download/v2.13/minimap2-2.13_x64-linux.tar.bz2 | tar -jxvf -
+./minimap2-2.13_x64-linux/minimap2
+```
+If you want to compile from the source, you need to have a C compiler, GNU make
+and zlib development files installed. Then type `make` in the source code
+directory to compile. If you see compilation errors, try `make sse2only=1`
+to disable SSE4 code, which will make minimap2 slightly slower.
 
-For modern x86-64 CPUs, just type `make` in the source code directory. This
-will compile a binary `minimap2` which you can copy to your desired location.
-If you see compilation errors, try `make sse2only=1` to disable SSE4. Minimap2
-will run a little slower. At present, minimap2 does not work with non-x86 CPUs
-or ancient CPUs that do not support SSE2. SSE2 is critical to the performance
-of minimap2.
+Minimap2 also works with ARM CPUs supporting the NEON instruction sets. To
+compile for 32 bit ARM architectures (such as ARMv7), use `make arm_neon=1`. To compile for for 64 bit ARM architectures (such as ARMv8), use `make arm_neon=1 aarch64=1`.
 
-## Algorithm Overview
+### <a name="general"></a>General usage
+
+Without any options, minimap2 takes a reference database and a query sequence
+file as input and produce approximate mapping, without base-level alignment
+(i.e. no CIGAR), in the [PAF format][paf]:
+```sh
+minimap2 ref.fa query.fq > approx-mapping.paf
+```
+You can ask minimap2 to generate CIGAR at the `cg` tag of PAF with:
+```sh
+minimap2 -c ref.fa query.fq > alignment.paf
+```
+or to output alignments in the [SAM format][sam]:
+```sh
+minimap2 -a ref.fa query.fq > alignment.sam
+```
+Minimap2 seamlessly works with gzip'd FASTA and FASTQ formats as input. You
+don't need to convert between FASTA and FASTQ or decompress gzip'd files first.
+
+For the human reference genome, minimap2 takes a few minutes to generate a
+minimizer index for the reference before mapping. To reduce indexing time, you
+can optionally save the index with option **-d** and replace the reference
+sequence file with the index file on the minimap2 command line:
+```sh
+minimap2 -d ref.mmi ref.fa                     # indexing
+minimap2 -a ref.mmi reads.fq > alignment.sam   # alignment
+```
+***Importantly***, it should be noted that once you build the index, indexing
+parameters such as **-k**, **-w**, **-H** and **-I** can't be changed during
+mapping. If you are running minimap2 for different data types, you will
+probably need to keep multiple indexes generated with different parameters.
+This makes minimap2 different from BWA which always uses the same index
+regardless of query data types.
+
+### <a name="cases"></a>Use cases
+
+Minimap2 uses the same base algorithm for all applications. However, due to the
+different data types it supports (e.g. short vs long reads; DNA vs mRNA reads),
+minimap2 needs to be tuned for optimal performance and accuracy. It is usually
+recommended to choose a preset with option **-x**, which sets multiple
+parameters at the same time. The default setting is the same as `map-ont`.
+
+#### <a name="map-long-genomic"></a>Map long noisy genomic reads
+
+```sh
+minimap2 -ax map-pb  ref.fa pacbio-reads.fq > aln.sam   # for PacBio subreads
+minimap2 -ax map-ont ref.fa ont-reads.fq > aln.sam      # for Oxford Nanopore reads
+```
+The difference between `map-pb` and `map-ont` is that `map-pb` uses
+homopolymer-compressed (HPC) minimizers as seeds, while `map-ont` uses ordinary
+minimizers as seeds. Emperical evaluation suggests HPC minimizers improve
+performance and sensitivity when aligning PacBio reads, but hurt when aligning
+Nanopore reads.
+
+#### <a name="map-long-splice"></a>Map long mRNA/cDNA reads
+
+```sh
+minimap2 -ax splice -uf -C5 ref.fa iso-seq.fq > aln.sam      # PacBio Iso-seq/traditional cDNA
+minimap2 -ax splice ref.fa nanopore-cdna.fa > aln.sam        # Nanopore 2D cDNA-seq
+minimap2 -ax splice -uf -k14 ref.fa direct-rna.fq > aln.sam  # Nanopore Direct RNA-seq
+minimap2 -ax splice --splice-flank=no SIRV.fa SIRV-seq.fa    # mapping against SIRV control
+```
+There are different long-read RNA-seq technologies, including tranditional
+full-length cDNA, EST, PacBio Iso-seq, Nanopore 2D cDNA-seq and Direct RNA-seq.
+They produce data of varying quality and properties. By default, `-x splice`
+assumes the read orientation relative to the transcript strand is unknown. It
+tries two rounds of alignment to infer the orientation and write the strand to
+the `ts` SAM/PAF tag if possible. For Iso-seq, Direct RNA-seq and tranditional
+full-length cDNAs, it would be desired to apply `-u f` to force minimap2 to
+consider the forward transcript strand only. This speeds up alignment with
+slight improvement to accuracy. For noisy Nanopore Direct RNA-seq reads, it is
+recommended to use a smaller k-mer size for increased sensitivity to the first
+or the last exons.
+
+Minimap2 rates an alignment by the score of the max-scoring sub-segment,
+*excluding* introns, and marks the best alignment as primary in SAM. When a
+spliced gene also has unspliced pseudogenes, minimap2 does not intentionally
+prefer spliced alignment, though in practice it more often marks the spliced
+alignment as the primary. By default, minimap2 outputs up to five secondary
+alignments (i.e. likely pseudogenes in the context of RNA-seq mapping). This
+can be tuned with option **-N**.
+
+For long RNA-seq reads, minimap2 may produce chimeric alignments potentially
+caused by gene fusions/structural variations or by an intron longer than the
+max intron length **-G** (200k by default). For now, it is not recommended to
+apply an excessively large **-G** as this slows down minimap2 and sometimes
+leads to false alignments.
+
+It is worth noting that by default `-x splice` prefers GT[A/G]..[C/T]AG
+over GT[C/T]..[A/G]AG, and then over other splicing signals. Considering
+one additional base improves the junction accuracy for noisy reads, but
+reduces the accuracy when aligning against the widely used SIRV control data.
+This is because SIRV does not honor the evolutionarily conservative splicing
+signal. If you are studying SIRV, you may apply `--splice-flank=no` to let
+minimap2 only model GT..AG, ignoring the additional base.
+
+#### <a name="long-overlap"></a>Find overlaps between long reads
+
+```sh
+minimap2 -x ava-pb  reads.fq reads.fq > ovlp.paf    # PacBio read overlap
+minimap2 -x ava-ont reads.fq reads.fq > ovlp.paf    # Oxford Nanopore read overlap
+```
+Similarly, `ava-pb` uses HPC minimizers while `ava-ont` uses ordinary
+minimizers. It is usually not recommended to perform base-level alignment in
+the overlapping mode because it is slow and may produce false positive
+overlaps. However, if performance is not a concern, you may try to add `-a` or
+`-c` anyway.
+
+#### <a name="short-genomic"></a>Map short accurate genomic reads
+
+```sh
+minimap2 -ax sr ref.fa reads-se.fq > aln.sam           # single-end alignment
+minimap2 -ax sr ref.fa read1.fq read2.fq > aln.sam     # paired-end alignment
+minimap2 -ax sr ref.fa reads-interleaved.fq > aln.sam  # paired-end alignment
+```
+When two read files are specified, minimap2 reads from each file in turn and
+merge them into an interleaved stream internally. Two reads are considered to
+be paired if they are adjacent in the input stream and have the same name (with
+the `/[0-9]` suffix trimmed if present). Single- and paired-end reads can be
+mixed.
+
+Minimap2 does not work well with short spliced reads. There are many capable
+RNA-seq mappers for short reads.
+
+#### <a name="full-genome"></a>Full genome/assembly alignment
+
+```sh
+minimap2 -ax asm5 ref.fa asm.fa > aln.sam       # assembly to assembly/ref alignment
+```
+For cross-species full-genome alignment, the scoring system needs to be tuned
+according to the sequence divergence.
+
+### <a name="advanced"></a>Advanced features
+
+#### <a name="long-cigar"></a>Working with >65535 CIGAR operations
+
+Due to a design flaw, BAM does not work with CIGAR strings with >65535
+operations (SAM and CRAM work). However, for ultra-long nanopore reads minimap2
+may align ~1% of read bases with long CIGARs beyond the capability of BAM. If
+you convert such SAM/CRAM to BAM, Picard and recent samtools will throw an
+error and abort. Older samtools and other tools may create corrupted BAM.
+
+To avoid this issue, you can add option `-L` at the minimap2 command line.
+This option moves a long CIGAR to the `CG` tag and leaves a fully clipped CIGAR
+at the SAM CIGAR column. Current tools that don't read CIGAR (e.g. merging and
+sorting) still work with such BAM records; tools that read CIGAR will
+effectively ignore these records. It has been decided that future tools will
+will seamlessly recognize long-cigar records generated by option `-L`.
+
+**TL;DR**: if you work with ultra-long reads and use tools that only process
+BAM files, please add option `-L`.
+
+#### <a name="cs"></a>The cs optional tag
+
+The `cs` SAM/PAF tag encodes bases at mismatches and INDELs. It matches regular
+expression `/(:[0-9]+|\*[a-z][a-z]|[=\+\-][A-Za-z]+)+/`. Like CIGAR, `cs`
+consists of series of operations.  Each leading character specifies the
+operation; the following sequence is the one involved in the operation.
+
+The `cs` tag is enabled by command line option `--cs`. The following alignment,
+for example:
+```txt
+CGATCGATAAATAGAGTAG---GAATAGCA
+||||||   ||||||||||   |||| |||
+CGATCG---AATAGAGTAGGTCGAATtGCA
+```
+is represented as `:6-ata:10+gtc:4*at:3`, where `:[0-9]+` represents an
+identical block, `-ata` represents a deltion, `+gtc` an insertion and `*at`
+indicates reference base `a` is substituted with a query base `t`. It is
+similar to the `MD` SAM tag but is standalone and easier to parse.
+
+If `--cs=long` is used, the `cs` string also contains identical sequences in
+the alignment. The above example will become
+`=CGATCG-ata=AATAGAGTAG+gtc=GAAT*at=GCA`. The long form of `cs` encodes both
+reference and query sequences in one string. The `cs` tag also encodes intron
+positions and splicing signals (see the [minimap2 manpage][manpage-cs] for
+details).
+
+#### <a name="paftools"></a>Working with the PAF format
+
+Minimap2 also comes with a (java)script [paftools.js](misc/paftools.js) that
+processes alignments in the PAF format. It calls variants from
+assembly-to-reference alignment, lifts over BED files based on alignment,
+converts between formats and provides utilities for various evaluations. For
+details, please see [misc/README.md](misc/README.md).
+
+### <a name="algo"></a>Algorithm overview
 
 In the following, minimap2 command line options have a dash ahead and are
-highlighted in bold.
+highlighted in bold. The description may help to tune minimap2 parameters.
 
 1. Read **-I** [=*4G*] reference bases, extract (**-k**,**-w**)-minimizers and
    index them in a hash table.
@@ -89,20 +312,48 @@ highlighted in bold.
 9. If there are more reference sequences, reopen the query file from the start
    and go to step 1; otherwise stop.
 
-## Limitations
+### <a name="help"></a>Getting help
+
+Manpage [minimap2.1][manpage] provides detailed description of minimap2
+command line options and optional tags. If you encounter bugs or have further
+questions or requests, you can raise an issue at the [issue page][issue].
+There is not a specific mailing list for the time being.
+
+### <a name="cite"></a>Citing minimap2
+
+If you use minimap2 in your work, please cite:
+
+> Li, H. (2018). Minimap2: pairwise alignment for nucleotide sequences.
+> Bioinformatics. [doi:10.1093/bioinformatics/bty191][doi]
+
+## <a name="dguide"></a>Developers' Guide
+
+Minimap2 is not only a command line tool, but also a programming library.
+It provides C APIs to build/load index and to align sequences against the
+index. File [example.c](example.c) demonstrates typical uses of C APIs. Header
+file [minimap.h](minimap.h) gives more detailed API documentation. Minimap2
+aims to keep APIs in this header stable. File [mmpriv.h](mmpriv.h) contains
+additional private APIs which may be subjected to changes frequently.
+
+This repository also provides Python bindings to a subset of C APIs. File
+[python/README.rst](python/README.rst) gives the full documentation;
+[python/minimap2.py](python/minimap2.py) shows an example. This Python
+extension, mappy, is also [available from PyPI][mappypypi] via `pip install
+mappy` or [from BioConda][mappyconda] via `conda install -c bioconda mappy`.
+
+## <a name="limit"></a>Limitations
 
 * Minimap2 may produce suboptimal alignments through long low-complexity
   regions where seed positions may be suboptimal. This should not be a big
   concern because even the optimal alignment may be wrong in such regions.
 
-* Minimap2 does not work well with Illumina short reads as of now.
+* Minimap2 requires SSE2 instructions on x86 CPUs or NEON on ARM CPUs. It is
+  possible to add non-SIMD support, but it would make minimap2 slower by
+  several times.
 
-* Minimap2 requires SSE2 instructions to compile. It is possible to add
-  non-SSE2 support, but it would make minimap2 slower by several times.
-
-In general, minimap2 is a young project with most code written since June, 2017.
-It may have bugs and room for improvements. Bug reports and suggestions are
-warmly welcomed.
+* Minimap2 does not work with a single query or database sequence ~2
+  billion bases or longer (2,147,483,647 to be exact). The total length of all
+  sequences can well exceed this threshold.
 
 
 
@@ -113,3 +364,12 @@ warmly welcomed.
 [longislnd]: https://www.ncbi.nlm.nih.gov/pubmed/27667791
 [gaba]: https://github.com/ocxtal/libgaba
 [ksw2]: https://github.com/lh3/ksw2
+[preprint]: https://arxiv.org/abs/1708.01492
+[release]: https://github.com/lh3/minimap2/releases
+[mappypypi]: https://pypi.python.org/pypi/mappy
+[mappyconda]: https://anaconda.org/bioconda/mappy
+[issue]: https://github.com/lh3/minimap2/issues
+[k8]: https://github.com/attractivechaos/k8
+[manpage]: https://lh3.github.io/minimap2/minimap2.html
+[manpage-cs]: https://lh3.github.io/minimap2/minimap2.html#10
+[doi]: https://doi.org/10.1093/bioinformatics/bty191

align.c

@@ -1,21 +1,24 @@
 #include <assert.h>
 #include <string.h>
+#include <stdlib.h>
+#include <math.h>
 #include "minimap.h"
 #include "mmpriv.h"
 #include "ksw2.h"
 
-static void ksw_gen_simple_mat(int m, int8_t *mat, int8_t a, int8_t b)
+static void ksw_gen_simple_mat(int m, int8_t *mat, int8_t a, int8_t b, int8_t sc_ambi)
 {
 	int i, j;
 	a = a < 0? -a : a;
 	b = b > 0? -b : b;
+	sc_ambi = sc_ambi > 0? -sc_ambi : sc_ambi;
 	for (i = 0; i < m - 1; ++i) {
 		for (j = 0; j < m - 1; ++j)
 			mat[i * m + j] = i == j? a : b;
-		mat[i * m + m - 1] = 0;
+		mat[i * m + m - 1] = sc_ambi;
 	}
 	for (j = 0; j < m; ++j)
-		mat[(m - 1) * m + j] = 0;
+		mat[(m - 1) * m + j] = sc_ambi;
 }
 
 static inline void mm_seq_rev(uint32_t len, uint8_t *seq)
@@ -26,86 +29,169 @@ static inline void mm_seq_rev(uint32_t len, uint8_t *seq)
 		t = seq[i], seq[i] = seq[len - 1 - i], seq[len - 1 - i] = t;
 }
 
-static inline int test_zdrop_aux(int32_t score, int i, int j, int32_t *max, int *max_i, int *max_j, int e, int zdrop)
+static inline void update_max_zdrop(int32_t score, int i, int j, int32_t *max, int *max_i, int *max_j, int e, int *max_zdrop, int pos[2][2])
 {
 	if (score < *max) {
 		int li = i - *max_i;
 		int lj = j - *max_j;
 		int diff = li > lj? li - lj : lj - li;
-		if (*max - score > zdrop + diff * e)
-			return 1;
+		int z = *max - score - diff * e;
+		if (z > *max_zdrop) {
+			*max_zdrop = z;
+			pos[0][0] = *max_i, pos[0][1] = i + 1;
+			pos[1][0] = *max_j, pos[1][1] = j + 1;
+		}
 	} else *max = score, *max_i = i, *max_j = j;
-	return 0;
 }
 
-static int mm_check_zdrop(const uint8_t *qseq, const uint8_t *tseq, uint32_t n_cigar, uint32_t *cigar, const int8_t *mat, int8_t q, int8_t e, int zdrop)
+static int mm_test_zdrop(void *km, const mm_mapopt_t *opt, const uint8_t *qseq, const uint8_t *tseq, uint32_t n_cigar, uint32_t *cigar, const int8_t *mat)
 {
 	uint32_t k;
-	int32_t score = 0, max = 0, max_i = -1, max_j = -1, i = 0, j = 0;
-	for (k = 0; k < n_cigar; ++k) {
+	int32_t score = 0, max = INT32_MIN, max_i = -1, max_j = -1, i = 0, j = 0, max_zdrop = 0;
+	int pos[2][2] = {{-1, -1}, {-1, -1}}, q_len, t_len;
+
+	// find the score and the region where score drops most along diagonal
+	for (k = 0, score = 0; k < n_cigar; ++k) {
 		uint32_t l, op = cigar[k]&0xf, len = cigar[k]>>4;
 		if (op == 0) {
 			for (l = 0; l < len; ++l) {
 				score += mat[tseq[i + l] * 5 + qseq[j + l]];
-				if (test_zdrop_aux(score, i+l, j+l, &max, &max_i, &max_j, e, zdrop)) return 1;
+				update_max_zdrop(score, i+l, j+l, &max, &max_i, &max_j, opt->e, &max_zdrop, pos);
 			}
 			i += len, j += len;
-		} else if (op == 1) {
-			score -= q + e * len, j += len;
-			if (test_zdrop_aux(score, i, j, &max, &max_i, &max_j, e, zdrop)) return 1;
-		} else if (op == 2 || op == 3) {
-			score -= q + e * len, i += len;
-			if (test_zdrop_aux(score, i, j, &max, &max_i, &max_j, e, zdrop)) return 1;
+		} else if (op == 1 || op == 2 || op == 3) {
+			score -= opt->q + opt->e * len;
+			if (op == 1) j += len; // insertion
+			else i += len;         // deletion
+			update_max_zdrop(score, i, j, &max, &max_i, &max_j, opt->e, &max_zdrop, pos);
 		}
 	}
-	return 0;
+
+	// test if there is an inversion in the most dropped region
+	q_len = pos[1][1] - pos[1][0], t_len = pos[0][1] - pos[0][0];
+	if (!(opt->flag&(MM_F_SPLICE|MM_F_SR|MM_F_FOR_ONLY|MM_F_REV_ONLY)) && max_zdrop > opt->zdrop_inv && q_len < opt->max_gap && t_len < opt->max_gap) {
+		uint8_t *qseq2;
+		void *qp;
+		int q_off, t_off;
+		qseq2 = (uint8_t*)kmalloc(km, q_len);
+		for (i = 0; i < q_len; ++i) {
+			int c = qseq[pos[1][1] - i - 1];
+			qseq2[i] = c >= 4? 4 : 3 - c;
+		}
+		qp = ksw_ll_qinit(km, 2, q_len, qseq2, 5, mat);
+		score = ksw_ll_i16(qp, t_len, tseq + pos[0][0], opt->q, opt->e, &q_off, &t_off);
+		kfree(km, qseq2);
+		kfree(km, qp);
+		if (score >= opt->min_chain_score * opt->a && score >= opt->min_dp_max)
+			return 2; // there is a potential inversion
+	}
+	return max_zdrop > opt->zdrop? 1 : 0;
 }
 
-static void mm_update_extra(mm_extra_t *p, const uint8_t *qseq, const uint8_t *tseq, const int8_t *mat, int8_t q, int8_t e)
+static void mm_fix_cigar(mm_reg1_t *r, const uint8_t *qseq, const uint8_t *tseq, int *qshift, int *tshift)
 {
-	uint32_t k, l, toff = 0, qoff = 0;
-	int32_t s = 0, max = 0, n_gtag = 0, n_ctac = 0;
+	mm_extra_t *p = r->p;
+	int32_t toff = 0, qoff = 0, to_shrink = 0;
+	uint32_t k;
+	*qshift = *tshift = 0;
+	if (p->n_cigar <= 1) return;
+	for (k = 0; k < p->n_cigar; ++k) { // indel left alignment
+		uint32_t op = p->cigar[k]&0xf, len = p->cigar[k]>>4;
+		if (len == 0) to_shrink = 1;
+		if (op == 0) {
+			toff += len, qoff += len;
+		} else if (op == 1 || op == 2) { // insertion or deletion
+			if (k > 0 && k < p->n_cigar - 1 && (p->cigar[k-1]&0xf) == 0 && (p->cigar[k+1]&0xf) == 0) {
+				int l, prev_len = p->cigar[k-1] >> 4;
+				if (op == 1) {
+					for (l = 0; l < prev_len; ++l)
+						if (qseq[qoff - 1 - l] != qseq[qoff + len - 1 - l])
+							break;
+				} else {
+					for (l = 0; l < prev_len; ++l)
+						if (tseq[toff - 1 - l] != tseq[toff + len - 1 - l])
+							break;
+				}
+				if (l > 0)
+					p->cigar[k-1] -= l<<4, p->cigar[k+1] += l<<4, qoff -= l, toff -= l;
+				if (l == prev_len) to_shrink = 1;
+			}
+			if (op == 1) qoff += len;
+			else toff += len;
+		} else if (op == 3) {
+			toff += len;
+		}
+	}
+	assert(qoff == r->qe - r->qs && toff == r->re - r->rs);
+	if (to_shrink) { // squeeze out zero-length operations
+		int32_t l = 0;
+		for (k = 0; k < p->n_cigar; ++k) // squeeze out zero-length operations
+			if (p->cigar[k]>>4 != 0)
+				p->cigar[l++] = p->cigar[k];
+		p->n_cigar = l;
+		for (k = l = 0; k < p->n_cigar; ++k) // merge two adjacent operations if they are the same
+			if (k == p->n_cigar - 1 || (p->cigar[k]&0xf) != (p->cigar[k+1]&0xf))
+				p->cigar[l++] = p->cigar[k];
+			else p->cigar[k+1] += p->cigar[k]>>4<<4; // add length to the next CIGAR operator
+		p->n_cigar = l;
+	}
+	if ((p->cigar[0]&0xf) == 1 || (p->cigar[0]&0xf) == 2) { // get rid of leading I or D
+		int32_t l = p->cigar[0] >> 4;
+		if ((p->cigar[0]&0xf) == 1) {
+			if (r->rev) r->qe -= l;
+			else r->qs += l;
+			*qshift = l;
+		} else r->rs += l, *tshift = l;
+		--p->n_cigar;
+		memmove(p->cigar, p->cigar + 1, p->n_cigar * 4);
+	}
+}
+
+static void mm_update_extra(mm_reg1_t *r, const uint8_t *qseq, const uint8_t *tseq, const int8_t *mat, int8_t q, int8_t e)
+{
+	uint32_t k, l;
+	int32_t s = 0, max = 0, qshift, tshift, toff = 0, qoff = 0;
+	mm_extra_t *p = r->p;
 	if (p == 0) return;
+	mm_fix_cigar(r, qseq, tseq, &qshift, &tshift);
+	qseq += qshift, tseq += tshift; // qseq and tseq may be shifted due to the removal of leading I/D
+	r->blen = r->mlen = 0;
 	for (k = 0; k < p->n_cigar; ++k) {
 		uint32_t op = p->cigar[k]&0xf, len = p->cigar[k]>>4;
 		if (op == 0) { // match/mismatch
+			int n_ambi = 0, n_diff = 0;
 			for (l = 0; l < len; ++l) {
 				int cq = qseq[qoff + l], ct = tseq[toff + l];
-				if (ct > 3 || cq > 3) ++p->n_ambi;
-				else if (ct != cq) ++p->n_diff;
+				if (ct > 3 || cq > 3) ++n_ambi;
+				else if (ct != cq) ++n_diff;
 				s += mat[ct * 5 + cq];
 				if (s < 0) s = 0;
 				else max = max > s? max : s;
 			}
-			toff += len, qoff += len, p->blen += len;
+			r->blen += len - n_ambi, r->mlen += len - (n_ambi + n_diff), p->n_ambi += n_ambi;
+			toff += len, qoff += len;
 		} else if (op == 1) { // insertion
 			int n_ambi = 0;
 			for (l = 0; l < len; ++l)
 				if (qseq[qoff + l] > 3) ++n_ambi;
-			qoff += len, p->blen += len;
-			p->n_ambi += n_ambi, p->n_diff += len - n_ambi;
+			r->blen += len - n_ambi, p->n_ambi += n_ambi;
 			s -= q + e * len;
 			if (s < 0) s = 0;
+			qoff += len;
 		} else if (op == 2) { // deletion
 			int n_ambi = 0;
 			for (l = 0; l < len; ++l)
 				if (tseq[toff + l] > 3) ++n_ambi;
-			toff += len, p->blen += len;
-			p->n_ambi += n_ambi, p->n_diff += len - n_ambi;
+			r->blen += len - n_ambi, p->n_ambi += n_ambi;
 			s -= q + e * len;
 			if (s < 0) s = 0;
+			toff += len;
 		} else if (op == 3) { // intron
-			uint8_t b[4];
-			b[0] = tseq[toff], b[1] = tseq[toff+1];
-			b[2] = tseq[toff+len-2], b[3] = tseq[toff+len-1];
-			if (memcmp(b, "\2\3\0\2", 4) == 0) ++n_gtag;
-			else if (memcmp(b, "\1\3\0\1", 4) == 0) ++n_ctac;
-			toff += len, p->blen += len;
+			toff += len;
 		}
 	}
 	p->dp_max = max;
-	if (n_gtag > n_ctac) p->trans_strand = 1;
-	else if (n_gtag < n_ctac) p->trans_strand = 2;
+	assert(qoff == r->qe - r->qs && toff == r->re - r->rs);
 }
 
 static void mm_append_cigar(mm_reg1_t *r, uint32_t n_cigar, uint32_t *cigar) // TODO: this calls the libc realloc()
@@ -113,12 +199,12 @@ static void mm_append_cigar(mm_reg1_t *r, uint32_t n_cigar, uint32_t *cigar) //
 	mm_extra_t *p;
 	if (n_cigar == 0) return;
 	if (r->p == 0) {
-		uint32_t capacity = n_cigar + sizeof(mm_extra_t);
+		uint32_t capacity = n_cigar + sizeof(mm_extra_t)/4;
 		kroundup32(capacity);
 		r->p = (mm_extra_t*)calloc(capacity, 4);
 		r->p->capacity = capacity;
-	} else if (r->p->n_cigar + n_cigar + sizeof(mm_extra_t) > r->p->capacity) {
-		r->p->capacity = r->p->n_cigar + n_cigar + sizeof(mm_extra_t);
+	} else if (r->p->n_cigar + n_cigar + sizeof(mm_extra_t)/4 > r->p->capacity) {
+		r->p->capacity = r->p->n_cigar + n_cigar + sizeof(mm_extra_t)/4;
 		kroundup32(r->p->capacity);
 		r->p = (mm_extra_t*)realloc(r->p, r->p->capacity * 4);
 	}
@@ -133,20 +219,100 @@ static void mm_append_cigar(mm_reg1_t *r, uint32_t n_cigar, uint32_t *cigar) //
 	}
 }
 
-static void mm_align_pair(void *km, const mm_mapopt_t *opt, int qlen, const uint8_t *qseq, int tlen, const uint8_t *tseq, const int8_t *mat, int w, int flag, ksw_extz_t *ez)
+static void mm_update_cigar_eqx(mm_reg1_t *r, const uint8_t *qseq, const uint8_t *tseq) // written by @armintoepfer
+{
+	uint32_t n_EQX = 0;
+	uint32_t k, l, m, cap, toff = 0, qoff = 0, n_M = 0;
+	mm_extra_t *p;
+	if (r->p == 0) return;
+	for (k = 0; k < r->p->n_cigar; ++k) {
+		uint32_t op = r->p->cigar[k]&0xf, len = r->p->cigar[k]>>4;
+		if (op == 0) {
+			while (len > 0) {
+				for (l = 0; l < len && qseq[qoff + l] == tseq[toff + l]; ++l) {} // run of "="; TODO: N<=>N is converted to "="
+				if (l > 0) { ++n_EQX; len -= l; toff += l; qoff += l; }
+
+				for (l = 0; l < len && qseq[qoff + l] != tseq[toff + l]; ++l) {} // run of "X"
+				if (l > 0) { ++n_EQX; len -= l; toff += l; qoff += l; }
+			}
+			++n_M;
+		} else if (op == 1) { // insertion
+			qoff += len;
+		} else if (op == 2) { // deletion
+			toff += len;
+		} else if (op == 3) { // intron
+			toff += len;
+		}
+	}
+	// update in-place if we can
+	if (n_EQX == n_M) {
+		for (k = 0; k < r->p->n_cigar; ++k) {
+			uint32_t op = r->p->cigar[k]&0xf, len = r->p->cigar[k]>>4;
+			if (op == 0) r->p->cigar[k] = len << 4 | 7;
+		}
+		return;
+	}
+	// allocate new storage
+	cap = r->p->n_cigar + (n_EQX - n_M) + sizeof(mm_extra_t);
+	kroundup32(cap);
+	p = (mm_extra_t*)calloc(cap, 4);
+	memcpy(p, r->p, sizeof(mm_extra_t));
+	p->capacity = cap;
+	// update cigar while copying
+	toff = qoff = m = 0;
+	for (k = 0; k < r->p->n_cigar; ++k) {
+		uint32_t op = r->p->cigar[k]&0xf, len = r->p->cigar[k]>>4;
+		if (op == 0) { // match/mismatch
+			while (len > 0) {
+				// match
+				for (l = 0; l < len && qseq[qoff + l] == tseq[toff + l]; ++l) {}
+				if (l > 0) p->cigar[m++] = l << 4 | 7;
+				len -= l;
+				toff += l, qoff += l;
+				// mismatch
+				for (l = 0; l < len && qseq[qoff + l] != tseq[toff + l]; ++l) {}
+				if (l > 0) p->cigar[m++] = l << 4 | 8;
+				len -= l;
+				toff += l, qoff += l;
+			}
+			continue;
+		} else if (op == 1) { // insertion
+			qoff += len;
+		} else if (op == 2) { // deletion
+			toff += len;
+		} else if (op == 3) { // intron
+			toff += len;
+		}
+		p->cigar[m++] = r->p->cigar[k];
+	}
+	p->n_cigar = m;
+	free(r->p);
+	r->p = p;
+}
+
+static void mm_align_pair(void *km, const mm_mapopt_t *opt, int qlen, const uint8_t *qseq, int tlen, const uint8_t *tseq, const int8_t *mat, int w, int end_bonus, int zdrop, int flag, ksw_extz_t *ez)
 {
 	if (mm_dbg_flag & MM_DBG_PRINT_ALN_SEQ) {
 		int i;
 		fprintf(stderr, "===> q=(%d,%d), e=(%d,%d), bw=%d, flag=%d, zdrop=%d <===\n", opt->q, opt->q2, opt->e, opt->e2, w, flag, opt->zdrop);
-		for (i = 0; i < tlen; ++i) fputc("ACGTN"[tseq[i]], stderr); fputc('\n', stderr);
-		for (i = 0; i < qlen; ++i) fputc("ACGTN"[qseq[i]], stderr); fputc('\n', stderr);
+		for (i = 0; i < tlen; ++i) fputc("ACGTN"[tseq[i]], stderr);
+		fputc('\n', stderr);
+		for (i = 0; i < qlen; ++i) fputc("ACGTN"[qseq[i]], stderr);
+		fputc('\n', stderr);
 	}
 	if (opt->flag & MM_F_SPLICE)
-		ksw_exts2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, opt->q2, opt->noncan, opt->zdrop, flag, ez);
+		ksw_exts2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, opt->q2, opt->noncan, zdrop, flag, ez);
 	else if (opt->q == opt->q2 && opt->e == opt->e2)
-		ksw_extz2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, w, opt->zdrop, flag, ez);
+		ksw_extz2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, w, zdrop, end_bonus, flag, ez);
 	else
-		ksw_extd2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, opt->q2, opt->e2, w, opt->zdrop, flag, ez);
+		ksw_extd2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, opt->q2, opt->e2, w, zdrop, end_bonus, flag, ez);
+	if (mm_dbg_flag & MM_DBG_PRINT_ALN_SEQ) {
+		int i;
+		fprintf(stderr, "score=%d, cigar=", ez->score);
+		for (i = 0; i < ez->n_cigar; ++i)
+			fprintf(stderr, "%d%c", ez->cigar[i]>>4, "MIDN"[ez->cigar[i]&0xf]);
+		fprintf(stderr, "\n");
+	}
 }
 
 static inline int mm_get_hplen_back(const mm_idx_t *mi, uint32_t rid, uint32_t x)
@@ -160,7 +326,7 @@ static inline int mm_get_hplen_back(const mm_idx_t *mi, uint32_t rid, uint32_t x
 
 static inline void mm_adjust_minier(const mm_idx_t *mi, uint8_t *const qseq0[2], mm128_t *a, int32_t *r, int32_t *q)
 {
-	if (mi->is_hpc) {
+	if (mi->flag & MM_I_HPC) {
 		const uint8_t *qseq = qseq0[a->x>>63];
 		int i, c;
 		*q = (int32_t)a->y;
@@ -175,20 +341,30 @@ static inline void mm_adjust_minier(const mm_idx_t *mi, uint8_t *const qseq0[2],
 	}
 }
 
-static void mm_filter_bad_seeds(void *km, int as1, int cnt1, mm128_t *a, int min_gap, int diff_thres, int max_ext_len, int max_ext_cnt)
+static int *collect_long_gaps(void *km, int as1, int cnt1, mm128_t *a, int min_gap, int *n_)
 {
-	int max_st, max_en, n, i, k, max, *K;
+	int i, n, *K;
+	*n_ = 0;
 	for (i = 1, n = 0; i < cnt1; ++i) { // count the number of gaps longer than min_gap
 		int gap = ((int32_t)a[as1 + i].y - a[as1 + i - 1].y) - ((int32_t)a[as1 + i].x - a[as1 + i - 1].x);
 		if (gap < -min_gap || gap > min_gap) ++n;
 	}
-	if (n <= 1) return;
+	if (n <= 1) return 0;
 	K = (int*)kmalloc(km, n * sizeof(int));
 	for (i = 1, n = 0; i < cnt1; ++i) { // store the positions of long gaps
 		int gap = ((int32_t)a[as1 + i].y - a[as1 + i - 1].y) - ((int32_t)a[as1 + i].x - a[as1 + i - 1].x);
 		if (gap < -min_gap || gap > min_gap)
 			K[n++] = i;
 	}
+	*n_ = n;
+	return K;
+}
+
+static void mm_filter_bad_seeds(void *km, int as1, int cnt1, mm128_t *a, int min_gap, int diff_thres, int max_ext_len, int max_ext_cnt)
+{
+	int max_st, max_en, n, i, k, max, *K;
+	K = collect_long_gaps(km, as1, cnt1, a, min_gap, &n);
+	if (K == 0) return;
 	max = 0, max_st = max_en = -1;
 	for (k = 0;; ++k) { // traverse long gaps
 		int gap, l, n_ins = 0, n_del = 0, qs, rs, max_diff = 0, max_diff_l = -1;
@@ -200,7 +376,7 @@ static void mm_filter_bad_seeds(void *km, int as1, int cnt1, mm128_t *a, int min
 			if (k == n) break;
 		}
 		i = K[k];
-		gap = ((int32_t)a[as1 + i].y - a[as1 + i - 1].y) - ((int32_t)a[as1 + i].x - a[as1 + i - 1].x);
+		gap = ((int32_t)a[as1 + i].y - (int32_t)a[as1 + i - 1].y) - (int32_t)(a[as1 + i].x - a[as1 + i - 1].x);
 		if (gap > 0) n_ins += gap;
 		else n_del += -gap;
 		qs = (int32_t)a[as1 + i - 1].y;
@@ -208,7 +384,7 @@ static void mm_filter_bad_seeds(void *km, int as1, int cnt1, mm128_t *a, int min
 		for (l = k + 1; l < n && l <= k + max_ext_cnt; ++l) {
 			int j = K[l], diff;
 			if ((int32_t)a[as1 + j].y - qs > max_ext_len || (int32_t)a[as1 + j].x - rs > max_ext_len) break;
-			gap = ((int32_t)a[as1 + j].y - (int32_t)a[as1 + j - 1].y) - (a[as1 + j].x - a[as1 + j - 1].x);
+			gap = ((int32_t)a[as1 + j].y - (int32_t)a[as1 + j - 1].y) - (int32_t)(a[as1 + j].x - a[as1 + j - 1].x);
 			if (gap > 0) n_ins += gap;
 			else n_del += -gap;
 			diff = n_ins + n_del - abs(n_ins - n_del);
@@ -221,38 +397,151 @@ static void mm_filter_bad_seeds(void *km, int as1, int cnt1, mm128_t *a, int min
 	kfree(km, K);
 }
 
-static void mm_fix_bad_ends(const mm_reg1_t *r, const mm128_t *a, int bw, int32_t *as, int32_t *cnt)
+static void mm_filter_bad_seeds_alt(void *km, int as1, int cnt1, mm128_t *a, int min_gap, int max_ext)
 {
-	int32_t i, l;
+	int n, k, *K;
+	K = collect_long_gaps(km, as1, cnt1, a, min_gap, &n);
+	if (K == 0) return;
+	for (k = 0; k < n;) {
+		int i = K[k], l;
+		int gap1 = ((int32_t)a[as1 + i].y - (int32_t)a[as1 + i - 1].y) - ((int32_t)a[as1 + i].x - (int32_t)a[as1 + i - 1].x);
+		int re1 = (int32_t)a[as1 + i].x;
+		int qe1 = (int32_t)a[as1 + i].y;
+		gap1 = gap1 > 0? gap1 : -gap1;
+		for (l = k + 1; l < n; ++l) {
+			int j = K[l], gap2, q_span_pre, rs2, qs2, m;
+			if ((int32_t)a[as1 + j].y - qe1 > max_ext || (int32_t)a[as1 + j].x - re1 > max_ext) break;
+			gap2 = ((int32_t)a[as1 + j].y - (int32_t)a[as1 + j - 1].y) - (int32_t)(a[as1 + j].x - a[as1 + j - 1].x);
+			q_span_pre = a[as1 + j - 1].y >> 32 & 0xff;
+			rs2 = (int32_t)a[as1 + j - 1].x + q_span_pre;
+			qs2 = (int32_t)a[as1 + j - 1].x + q_span_pre;
+			m = rs2 - re1 < qs2 - qe1? rs2 - re1 : qs2 - qe1;
+			gap2 = gap2 > 0? gap2 : -gap2;
+			if (m > gap1 + gap2) break;
+			re1 = (int32_t)a[as1 + j].x;
+			qe1 = (int32_t)a[as1 + j].y;
+			gap1 = gap2;
+		}
+		if (l > k + 1) {
+			int j, end = K[l - 1];
+			for (j = K[k]; j < end; ++j)
+				a[as1 + j].y |= MM_SEED_IGNORE;
+			a[as1 + end].y |= MM_SEED_LONG_JOIN;
+		}
+		k = l;
+	}
+	kfree(km, K);
+}
+
+static void mm_fix_bad_ends(const mm_reg1_t *r, const mm128_t *a, int bw, int min_match, int32_t *as, int32_t *cnt)
+{
+	int32_t i, l, m;
 	*as = r->as, *cnt = r->cnt;
 	if (r->cnt < 3) return;
-	l = a[r->as].y >> 32 & 0xff;
+	m = l = a[r->as].y >> 32 & 0xff;
 	for (i = r->as + 1; i < r->as + r->cnt - 1; ++i) {
 		int32_t lq, lr, min, max;
+		int32_t q_span = a[i].y >> 32 & 0xff;
+		if (a[i].y & MM_SEED_LONG_JOIN) break;
 		lr = (int32_t)a[i].x - (int32_t)a[i-1].x;
 		lq = (int32_t)a[i].y - (int32_t)a[i-1].y;
 		min = lr < lq? lr : lq;
 		max = lr > lq? lr : lq;
 		if (max - min > l >> 1) *as = i;
 		l += min;
-		if (l >= bw << 1) break;
+		m += min < q_span? min : q_span;
+		if (l >= bw << 1 || (m >= min_match && m >= bw) || m >= r->mlen >> 1) break;
 	}
 	*cnt = r->as + r->cnt - *as;
-	l = a[r->as + r->cnt - 1].y >> 32 & 0xff;
+	m = l = a[r->as + r->cnt - 1].y >> 32 & 0xff;
 	for (i = r->as + r->cnt - 2; i > *as; --i) {
 		int32_t lq, lr, min, max;
+		int32_t q_span = a[i+1].y >> 32 & 0xff;
+		if (a[i+1].y & MM_SEED_LONG_JOIN) break;
 		lr = (int32_t)a[i+1].x - (int32_t)a[i].x;
 		lq = (int32_t)a[i+1].y - (int32_t)a[i].y;
 		min = lr < lq? lr : lq;
 		max = lr > lq? lr : lq;
 		if (max - min > l >> 1) *cnt = i + 1 - *as;
 		l += min;
-		if (l >= bw) break;
+		m += min < q_span? min : q_span;
+		if (l >= bw << 1 || (m >= min_match && m >= bw) || m >= r->mlen >> 1) break;
 	}
 }
 
-static void mm_align1(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int qlen, uint8_t *qseq0[2], mm_reg1_t *r, mm_reg1_t *r2, mm128_t *a, ksw_extz_t *ez, int splice_flag)
+static void mm_max_stretch(const mm_reg1_t *r, const mm128_t *a, int32_t *as, int32_t *cnt)
 {
+	int32_t i, score, max_score, len, max_i, max_len;
+
+	*as = r->as, *cnt = r->cnt;
+	if (r->cnt < 2) return;
+
+	max_score = -1, max_i = -1, max_len = 0;
+	score = a[r->as].y >> 32 & 0xff, len = 1;
+	for (i = r->as + 1; i < r->as + r->cnt; ++i) {
+		int32_t lq, lr, q_span;
+		q_span = a[i].y >> 32 & 0xff;
+		lr = (int32_t)a[i].x - (int32_t)a[i-1].x;
+		lq = (int32_t)a[i].y - (int32_t)a[i-1].y;
+		if (lq == lr) {
+			score += lq < q_span? lq : q_span;
+			++len;
+		} else {
+			if (score > max_score)
+				max_score = score, max_len = len, max_i = i - len;
+			score = q_span, len = 1;
+		}
+	}
+	if (score > max_score)
+		max_score = score, max_len = len, max_i = i - len;
+	*as = max_i, *cnt = max_len;
+}
+
+static int mm_seed_ext_score(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, const int8_t mat[25], int qlen, uint8_t *qseq0[2], const mm128_t *a)
+{
+	uint8_t *qseq, *tseq;
+	int q_span = a->y>>32&0xff, qs, qe, rs, re, rid, score, q_off, t_off, ext_len = opt->anchor_ext_len;
+	void *qp;
+	rid = a->x<<1>>33;
+	re = (uint32_t)a->x + 1, rs = re - q_span;
+	qe = (uint32_t)a->y + 1, qs = qe - q_span;
+	rs = rs - ext_len > 0? rs - ext_len : 0;
+	qs = qs - ext_len > 0? qs - ext_len : 0;
+	re = re + ext_len < (int32_t)mi->seq[rid].len? re + ext_len : mi->seq[rid].len;
+	qe = qe + ext_len < qlen? qe + ext_len : qlen;
+	tseq = (uint8_t*)kmalloc(km, re - rs);
+	mm_idx_getseq(mi, rid, rs, re, tseq);
+	qseq = qseq0[a->x>>63] + qs;
+	qp = ksw_ll_qinit(km, 2, qe - qs, qseq, 5, mat);
+	score = ksw_ll_i16(qp, re - rs, tseq, opt->q, opt->e, &q_off, &t_off);
+	kfree(km, tseq);
+	kfree(km, qp);
+	return score;
+}
+
+static void mm_fix_bad_ends_splice(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, const mm_reg1_t *r, const int8_t mat[25], int qlen, uint8_t *qseq0[2], const mm128_t *a, int *as1, int *cnt1)
+{ // this assumes a very crude k-mer based mode; it is not necessary to use a good model just for filtering bounary exons
+	int score;
+	double log_gap;
+	*as1 = r->as, *cnt1 = r->cnt;
+	if (r->cnt < 3) return;
+	log_gap = log((int32_t)a[r->as + 1].x - (int32_t)a[r->as].x);
+	if ((a[r->as].y>>32&0xff) < log_gap + opt->anchor_ext_shift) {
+		score = mm_seed_ext_score(km, opt, mi, mat, qlen, qseq0, &a[r->as]);
+		if ((double)score / mat[0] < log_gap + opt->anchor_ext_shift) // a more exact format is "score < log_4(gap) + shift"
+			++(*as1), --(*cnt1);
+	}
+	log_gap = log((int32_t)a[r->as + r->cnt - 1].x - (int32_t)a[r->as + r->cnt - 2].x);
+	if ((a[r->as + r->cnt - 1].y>>32&0xff) < log_gap + opt->anchor_ext_shift) {
+		score = mm_seed_ext_score(km, opt, mi, mat, qlen, qseq0, &a[r->as + r->cnt - 1]);
+		if ((double)score / mat[0] < log_gap + opt->anchor_ext_shift)
+			--(*cnt1);
+	}
+}
+
+static void mm_align1(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int qlen, uint8_t *qseq0[2], mm_reg1_t *r, mm_reg1_t *r2, int n_a, mm128_t *a, ksw_extz_t *ez, int splice_flag)
+{
+	int is_sr = !!(opt->flag & MM_F_SR), is_splice = !!(opt->flag & MM_F_SPLICE);
 	int32_t rid = a[r->as].x<<1>>33, rev = a[r->as].x>>63, as1, cnt1;
 	uint8_t *tseq, *qseq;
 	int32_t i, l, bw, dropped = 0, extra_flag = 0, rs0, re0, qs0, qe0;
@@ -260,47 +549,114 @@ static void mm_align1(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int
 	int32_t rs1, qs1, re1, qe1;
 	int8_t mat[25];
 
-	if (r->cnt == 0) return;
-	ksw_gen_simple_mat(5, mat, opt->a, opt->b);
-	bw = (int)(opt->bw * 1.5 + 1.);
+	if (is_sr) assert(!(mi->flag & MM_I_HPC)); // HPC won't work with SR because with HPC we can't easily tell if there is a gap
 
 	r2->cnt = 0;
-	if (!(opt->flag & MM_F_SPLICE))
-		mm_fix_bad_ends(r, a, opt->bw, &as1, &cnt1);
-	else as1 = r->as, cnt1 = r->cnt;
-	mm_filter_bad_seeds(km, as1, cnt1, a, 10, 40, opt->max_gap>>1, 10);
-	mm_adjust_minier(mi, qseq0, &a[as1], &rs, &qs);
-	mm_adjust_minier(mi, qseq0, &a[as1 + cnt1 - 1], &re, &qe);
+	if (r->cnt == 0) return;
+	ksw_gen_simple_mat(5, mat, opt->a, opt->b, opt->sc_ambi);
+	bw = (int)(opt->bw * 1.5 + 1.);
 
-	if (opt->flag & MM_F_SPLICE) {
+	if (is_sr && !(mi->flag & MM_I_HPC)) {
+		mm_max_stretch(r, a, &as1, &cnt1);
+		rs = (int32_t)a[as1].x + 1 - (int32_t)(a[as1].y>>32&0xff);
+		qs = (int32_t)a[as1].y + 1 - (int32_t)(a[as1].y>>32&0xff);
+		re = (int32_t)a[as1+cnt1-1].x + 1;
+		qe = (int32_t)a[as1+cnt1-1].y + 1;
+	} else {
+		if (!(opt->flag & MM_F_NO_END_FLT)) {
+			if (is_splice)
+				mm_fix_bad_ends_splice(km, opt, mi, r, mat, qlen, qseq0, a, &as1, &cnt1);
+			else
+				mm_fix_bad_ends(r, a, opt->bw, opt->min_chain_score * 2, &as1, &cnt1);
+		} else as1 = r->as, cnt1 = r->cnt;
+		mm_filter_bad_seeds(km, as1, cnt1, a, 10, 40, opt->max_gap>>1, 10);
+		mm_filter_bad_seeds_alt(km, as1, cnt1, a, 30, opt->max_gap>>1);
+		mm_adjust_minier(mi, qseq0, &a[as1], &rs, &qs);
+		mm_adjust_minier(mi, qseq0, &a[as1 + cnt1 - 1], &re, &qe);
+	}
+	assert(cnt1 > 0);
+
+	if (is_splice) {
 		if (splice_flag & MM_F_SPLICE_FOR) extra_flag |= rev? KSW_EZ_SPLICE_REV : KSW_EZ_SPLICE_FOR;
 		if (splice_flag & MM_F_SPLICE_REV) extra_flag |= rev? KSW_EZ_SPLICE_FOR : KSW_EZ_SPLICE_REV;
-		if (splice_flag & MM_F_SPLICE_BOTH) extra_flag |= KSW_EZ_SPLICE_FOR|KSW_EZ_SPLICE_REV;
+		if (opt->flag & MM_F_SPLICE_FLANK) extra_flag |= KSW_EZ_SPLICE_FLANK;
 	}
 
-	// compute rs0 and qs0
-	if (r->split && as1 > 0) {
-		mm_adjust_minier(mi, qseq0, &a[as1-1], &rs0, &qs0);
+	/* Look for the start and end of regions to perform DP. This sounds easy
+	 * but is in fact tricky. Excessively small regions lead to unnecessary
+	 * clippings and lose alignable sequences. Excessively large regions
+	 * occasionally lead to large overlaps between two chains and may cause
+	 * loss of alignments in corner cases. */
+	if (is_sr) {
+		qs0 = 0, qe0 = qlen;
+		l = qs;
+		l += l * opt->a + opt->end_bonus > opt->q? (l * opt->a + opt->end_bonus - opt->q) / opt->e : 0;
+		rs0 = rs - l > 0? rs - l : 0;
+		l = qlen - qe;
+		l += l * opt->a + opt->end_bonus > opt->q? (l * opt->a + opt->end_bonus - opt->q) / opt->e : 0;
+		re0 = re + l < (int32_t)mi->seq[rid].len? re + l : mi->seq[rid].len;
 	} else {
-		if (qs > 0 && rs > 0) { // actually this is always true
+		// compute rs0 and qs0
+		rs0 = (int32_t)a[r->as].x + 1 - (int32_t)(a[r->as].y>>32&0xff);
+		qs0 = (int32_t)a[r->as].y + 1 - (int32_t)(a[r->as].y>>32&0xff);
+		if (rs0 < 0) rs0 = 0; // this may happen when HPC is in use
+		assert(qs0 >= 0); // this should never happen, or it is logic error
+		rs1 = qs1 = 0;
+		for (i = r->as - 1, l = 0; i >= 0 && a[i].x>>32 == a[r->as].x>>32; --i) { // inspect nearby seeds
+			int32_t x = (int32_t)a[i].x + 1 - (int32_t)(a[i].y>>32&0xff);
+			int32_t y = (int32_t)a[i].y + 1 - (int32_t)(a[i].y>>32&0xff);
+			if (x < rs0 && y < qs0) {
+				if (++l > opt->min_cnt) {
+					l = rs0 - x > qs0 - y? rs0 - x : qs0 - y;
+					rs1 = rs0 - l, qs1 = qs0 - l;
+					break;
+				}
+			}
+		}
+		if (qs > 0 && rs > 0) {
 			l = qs < opt->max_gap? qs : opt->max_gap;
-			qs0 = qs - l;
+			qs1 = qs1 > qs - l? qs1 : qs - l;
+			qs0 = qs0 < qs1? qs0 : qs1; // at least include qs0
 			l += l * opt->a > opt->q? (l * opt->a - opt->q) / opt->e : 0;
 			l = l < opt->max_gap? l : opt->max_gap;
 			l = l < rs? l : rs;
-			rs0 = rs - l;
+			rs1 = rs1 > rs - l? rs1 : rs - l;
+			rs0 = rs0 < rs1? rs0 : rs1;
 		} else rs0 = rs, qs0 = qs;
-
+		// compute re0 and qe0
+		re0 = (int32_t)a[r->as + r->cnt - 1].x + 1;
+		qe0 = (int32_t)a[r->as + r->cnt - 1].y + 1;
+		re1 = mi->seq[rid].len, qe1 = qlen;
+		for (i = r->as + r->cnt, l = 0; i < n_a && a[i].x>>32 == a[r->as].x>>32; ++i) { // inspect nearby seeds
+			int32_t x = (int32_t)a[i].x + 1;
+			int32_t y = (int32_t)a[i].y + 1;
+			if (x > re0 && y > qe0) {
+				if (++l > opt->min_cnt) {
+					l = x - re0 > y - qe0? x - re0 : y - qe0;
+					re1 = re0 + l, qe1 = qe0 + l;
+					break;
+				}
+			}
+		}
+		if (qe < qlen && re < (int32_t)mi->seq[rid].len) {
+			l = qlen - qe < opt->max_gap? qlen - qe : opt->max_gap;
+			qe1 = qe1 < qe + l? qe1 : qe + l;
+			qe0 = qe0 > qe1? qe0 : qe1; // at least include qe0
+			l += l * opt->a > opt->q? (l * opt->a - opt->q) / opt->e : 0;
+			l = l < opt->max_gap? l : opt->max_gap;
+			l = l < (int32_t)mi->seq[rid].len - re? l : mi->seq[rid].len - re;
+			re1 = re1 < re + l? re1 : re + l;
+			re0 = re0 > re1? re0 : re1;
+		} else re0 = re, qe0 = qe;
+	}
+	if (a[r->as].y & MM_SEED_SELF) {
+		int max_ext = r->qs > r->rs? r->qs - r->rs : r->rs - r->qs;
+		if (r->rs - rs0 > max_ext) rs0 = r->rs - max_ext;
+		if (r->qs - qs0 > max_ext) qs0 = r->qs - max_ext;
+		max_ext = r->qe > r->re? r->qe - r->re : r->re - r->qe;
+		if (re0 - r->re > max_ext) re0 = r->re + max_ext;
+		if (qe0 - r->qe > max_ext) qe0 = r->qe + max_ext;
 	}
-	// compute re0 and qe0
-	if (qe < qlen && re < mi->seq[rid].len) {
-		l = qlen - qe < opt->max_gap? qlen - qe : opt->max_gap;
-		qe0 = qe + l;
-		l += l * opt->a > opt->q? (l * opt->a - opt->q) / opt->e : 0;
-		l = l < opt->max_gap? l : opt->max_gap;
-		l = l < mi->seq[rid].len - re? l : mi->seq[rid].len - re;
-		re0 = re + l;
-	} else re0 = re, qe0 = qe;
 
 	assert(re0 > rs0);
 	tseq = (uint8_t*)kmalloc(km, re0 - rs0);
@@ -310,44 +666,62 @@ static void mm_align1(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int
 		mm_idx_getseq(mi, rid, rs0, rs, tseq);
 		mm_seq_rev(qs - qs0, qseq);
 		mm_seq_rev(rs - rs0, tseq);
-		mm_align_pair(km, opt, qs - qs0, qseq, rs - rs0, tseq, mat, bw, extra_flag|KSW_EZ_EXTZ_ONLY|KSW_EZ_RIGHT|KSW_EZ_REV_CIGAR, ez);
+		mm_align_pair(km, opt, qs - qs0, qseq, rs - rs0, tseq, mat, bw, opt->end_bonus, r->split_inv? opt->zdrop_inv : opt->zdrop, extra_flag|KSW_EZ_EXTZ_ONLY|KSW_EZ_RIGHT|KSW_EZ_REV_CIGAR, ez);
 		if (ez->n_cigar > 0) {
 			mm_append_cigar(r, ez->n_cigar, ez->cigar);
 			r->p->dp_score += ez->max;
 		}
-		rs1 = rs - (ez->max_t + 1);
-		qs1 = qs - (ez->max_q + 1);
+		rs1 = rs - (ez->reach_end? ez->mqe_t + 1 : ez->max_t + 1);
+		qs1 = qs - (ez->reach_end? qs - qs0 : ez->max_q + 1);
 		mm_seq_rev(qs - qs0, qseq);
 	} else rs1 = rs, qs1 = qs;
 	re1 = rs, qe1 = qs;
 	assert(qs1 >= 0 && rs1 >= 0);
 
-	for (i = 1; i < cnt1; ++i) { // gap filling
+	for (i = is_sr? cnt1 - 1 : 1; i < cnt1; ++i) { // gap filling
 		if ((a[as1+i].y & (MM_SEED_IGNORE|MM_SEED_TANDEM)) && i != cnt1 - 1) continue;
-		mm_adjust_minier(mi, qseq0, &a[as1 + i], &re, &qe);
+		if (is_sr && !(mi->flag & MM_I_HPC)) {
+			re = (int32_t)a[as1 + i].x + 1;
+			qe = (int32_t)a[as1 + i].y + 1;
+		} else mm_adjust_minier(mi, qseq0, &a[as1 + i], &re, &qe);
 		re1 = re, qe1 = qe;
 		if (i == cnt1 - 1 || (a[as1+i].y&MM_SEED_LONG_JOIN) || (qe - qs >= opt->min_ksw_len && re - rs >= opt->min_ksw_len)) {
-			int bw1 = bw;
+			int j, bw1 = bw, zdrop_code;
 			if (a[as1+i].y & MM_SEED_LONG_JOIN)
 				bw1 = qe - qs > re - rs? qe - qs : re - rs;
+			// perform alignment
 			qseq = &qseq0[rev][qs];
 			mm_idx_getseq(mi, rid, rs, re, tseq);
-			mm_align_pair(km, opt, qe - qs, qseq, re - rs, tseq, mat, bw1, extra_flag|KSW_EZ_APPROX_MAX, ez);
-			if (mm_check_zdrop(qseq, tseq, ez->n_cigar, ez->cigar, mat, opt->q, opt->e, opt->zdrop))
-				mm_align_pair(km, opt, qe - qs, qseq, re - rs, tseq, mat, bw1, extra_flag, ez);
+			if (is_sr) { // perform ungapped alignment
+				assert(qe - qs == re - rs);
+				ksw_reset_extz(ez);
+				for (j = 0, ez->score = 0; j < qe - qs; ++j) {
+					if (qseq[j] >= 4 || tseq[j] >= 4) ez->score += opt->e2;
+					else ez->score += qseq[j] == tseq[j]? opt->a : -opt->b;
+				}
+				ez->cigar = ksw_push_cigar(km, &ez->n_cigar, &ez->m_cigar, ez->cigar, 0, qe - qs);
+			} else { // perform normal gapped alignment
+				mm_align_pair(km, opt, qe - qs, qseq, re - rs, tseq, mat, bw1, -1, opt->zdrop, extra_flag|KSW_EZ_APPROX_MAX, ez); // first pass: with approximate Z-drop
+			}
+			// test Z-drop and inversion Z-drop
+			if ((zdrop_code = mm_test_zdrop(km, opt, qseq, tseq, ez->n_cigar, ez->cigar, mat)) != 0)
+				mm_align_pair(km, opt, qe - qs, qseq, re - rs, tseq, mat, bw1, -1, zdrop_code == 2? opt->zdrop_inv : opt->zdrop, extra_flag, ez); // second pass: lift approximate
+			// update CIGAR
 			if (ez->n_cigar > 0)
 				mm_append_cigar(r, ez->n_cigar, ez->cigar);
 			if (ez->zdropped) { // truncated by Z-drop; TODO: sometimes Z-drop kicks in because the next seed placement is wrong. This can be fixed in principle.
-				int j;
 				for (j = i - 1; j >= 0; --j)
-					if ((int32_t)a[as1 + j].x < re + ez->max_t)
+					if ((int32_t)a[as1 + j].x <= rs + ez->max_t)
 						break;
 				dropped = 1;
+				if (j < 0) j = 0;
 				r->p->dp_score += ez->max;
 				re1 = rs + (ez->max_t + 1);
 				qe1 = qs + (ez->max_q + 1);
-				if (cnt1 - (j + 1) >= opt->min_cnt)
-					mm_split_reg(r, r2, j + 1, qlen, a);
+				if (cnt1 - (j + 1) >= opt->min_cnt) {
+					mm_split_reg(r, r2, as1 + j + 1 - r->as, qlen, a);
+					if (zdrop_code == 2) r2->split_inv = 1;
+				}
 				break;
 			} else r->p->dp_score += ez->score;
 			rs = re, qs = qe;
@@ -357,13 +731,13 @@ static void mm_align1(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int
 	if (!dropped && qe < qe0 && re < re0) { // right extension
 		qseq = &qseq0[rev][qe];
 		mm_idx_getseq(mi, rid, re, re0, tseq);
-		mm_align_pair(km, opt, qe0 - qe, qseq, re0 - re, tseq, mat, bw, extra_flag|KSW_EZ_EXTZ_ONLY, ez);
+		mm_align_pair(km, opt, qe0 - qe, qseq, re0 - re, tseq, mat, bw, opt->end_bonus, opt->zdrop, extra_flag|KSW_EZ_EXTZ_ONLY, ez);
 		if (ez->n_cigar > 0) {
 			mm_append_cigar(r, ez->n_cigar, ez->cigar);
 			r->p->dp_score += ez->max;
 		}
-		re1 = re + (ez->max_t + 1);
-		qe1 = qe + (ez->max_q + 1);
+		re1 = re + (ez->reach_end? ez->mqe_t + 1 : ez->max_t + 1);
+		qe1 = qe + (ez->reach_end? qe0 - qe : ez->max_q + 1);
 	}
 	assert(qe1 <= qlen);
 
@@ -374,7 +748,8 @@ static void mm_align1(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int
 	assert(re1 - rs1 <= re0 - rs0);
 	if (r->p) {
 		mm_idx_getseq(mi, rid, rs1, re1, tseq);
-		mm_update_extra(r->p, &qseq0[r->rev][qs1], tseq, mat, opt->q, opt->e);
+		mm_update_extra(r, &qseq0[r->rev][qs1], tseq, mat, opt->q, opt->e);
+		if (opt->flag & MM_F_EQX) mm_update_cigar_eqx(r, &qseq0[r->rev][qs1], tseq);
 		if (rev && r->p->trans_strand)
 			r->p->trans_strand ^= 3; // flip to the read strand
 	}
@@ -394,15 +769,15 @@ static int mm_align1_inv(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, i
 	if (r1->id != r1->parent && r1->parent != MM_PARENT_TMP_PRI) return 0;
 	if (r2->id != r2->parent && r2->parent != MM_PARENT_TMP_PRI) return 0;
 	if (r1->rid != r2->rid || r1->rev != r2->rev) return 0;
-	ql = r2->qs - r1->qe;
+	ql = r1->rev? r1->qs - r2->qe : r2->qs - r1->qe;
 	tl = r2->rs - r1->re;
 	if (ql < opt->min_chain_score || ql > opt->max_gap) return 0;
 	if (tl < opt->min_chain_score || tl > opt->max_gap) return 0;
 
-	ksw_gen_simple_mat(5, mat, opt->a, opt->b);
+	ksw_gen_simple_mat(5, mat, opt->a, opt->b, opt->sc_ambi);
 	tseq = (uint8_t*)kmalloc(km, tl);
 	mm_idx_getseq(mi, r1->rid, r1->re, r2->rs, tseq);
-	qseq = &qseq0[!r1->rev][qlen - r2->qs];
+	qseq = r1->rev? &qseq0[0][r2->qe] : &qseq0[1][qlen - r2->qs];
 
 	mm_seq_rev(ql, qseq);
 	mm_seq_rev(tl, tseq);
@@ -413,17 +788,27 @@ static int mm_align1_inv(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, i
 	mm_seq_rev(tl, tseq);
 	if (score < opt->min_dp_max) goto end_align1_inv;
 	q_off = ql - (q_off + 1), t_off = tl - (t_off + 1);
-	mm_align_pair(km, opt, ql - q_off, qseq + q_off, tl - t_off, tseq + t_off, mat, (int)(opt->bw * 1.5), KSW_EZ_EXTZ_ONLY, ez);
+	mm_align_pair(km, opt, ql - q_off, qseq + q_off, tl - t_off, tseq + t_off, mat, (int)(opt->bw * 1.5), -1, opt->zdrop, KSW_EZ_EXTZ_ONLY, ez);
 	if (ez->n_cigar == 0) goto end_align1_inv; // should never be here
 	mm_append_cigar(r_inv, ez->n_cigar, ez->cigar);
 	r_inv->p->dp_score = ez->max;
-	mm_update_extra(r_inv->p, qseq + q_off, tseq + t_off, mat, opt->q, opt->e);
 	r_inv->id = -1;
 	r_inv->parent = MM_PARENT_UNSET;
 	r_inv->inv = 1;
 	r_inv->rev = !r1->rev;
-	r_inv->qs = r1->qe + q_off, r_inv->qe = r_inv->qs + ez->max_q + 1;
-	r_inv->rs = r1->re + t_off, r_inv->re = r_inv->rs + ez->max_t + 1;
+	r_inv->rid = r1->rid;
+	r_inv->div = -1.0f;
+	if (r_inv->rev == 0) {
+		r_inv->qs = r2->qe + q_off;
+		r_inv->qe = r_inv->qs + ez->max_q + 1;
+	} else {
+		r_inv->qe = r2->qs - q_off;
+		r_inv->qs = r_inv->qe - (ez->max_q + 1);
+	}
+	r_inv->rs = r1->re + t_off;
+	r_inv->re = r_inv->rs + ez->max_t + 1;
+	mm_update_extra(r_inv, &qseq[q_off], &tseq[t_off], mat, opt->q, opt->e);
+	if (opt->flag & MM_F_EQX) mm_update_cigar_eqx(r_inv, &qseq[q_off], &tseq[t_off]);
 	ret = 1;
 end_align1_inv:
 	kfree(km, tseq);
@@ -443,28 +828,29 @@ static inline mm_reg1_t *mm_insert_reg(const mm_reg1_t *r, int i, int *n_regs, m
 mm_reg1_t *mm_align_skeleton(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int qlen, const char *qstr, int *n_regs_, mm_reg1_t *regs, mm128_t *a)
 {
 	extern unsigned char seq_nt4_table[256];
-	int32_t i, n_regs = *n_regs_;
+	int32_t i, n_regs = *n_regs_, n_a;
 	uint8_t *qseq0[2];
 	ksw_extz_t ez;
 
 	// encode the query sequence
-	qseq0[0] = (uint8_t*)kmalloc(km, qlen);
-	qseq0[1] = (uint8_t*)kmalloc(km, qlen);
+	qseq0[0] = (uint8_t*)kmalloc(km, qlen * 2);
+	qseq0[1] = qseq0[0] + qlen;
 	for (i = 0; i < qlen; ++i) {
 		qseq0[0][i] = seq_nt4_table[(uint8_t)qstr[i]];
 		qseq0[1][qlen - 1 - i] = qseq0[0][i] < 4? 3 - qseq0[0][i] : 4;
 	}
 
 	// align through seed hits
+	n_a = mm_squeeze_a(km, n_regs, regs, a);
 	memset(&ez, 0, sizeof(ksw_extz_t));
 	for (i = 0; i < n_regs; ++i) {
 		mm_reg1_t r2;
-		if ((opt->flag&MM_F_SPLICE) && (opt->flag&MM_F_SPLICE_FOR) && (opt->flag&MM_F_SPLICE_REV)) {
+		if ((opt->flag&MM_F_SPLICE) && (opt->flag&MM_F_SPLICE_FOR) && (opt->flag&MM_F_SPLICE_REV)) { // then do two rounds of alignments for both strands
 			mm_reg1_t s[2], s2[2];
 			int which, trans_strand;
 			s[0] = s[1] = regs[i];
-			mm_align1(km, opt, mi, qlen, qseq0, &s[0], &s2[0], a, &ez, MM_F_SPLICE_FOR);
-			mm_align1(km, opt, mi, qlen, qseq0, &s[1], &s2[1], a, &ez, MM_F_SPLICE_REV);
+			mm_align1(km, opt, mi, qlen, qseq0, &s[0], &s2[0], n_a, a, &ez, MM_F_SPLICE_FOR);
+			mm_align1(km, opt, mi, qlen, qseq0, &s[1], &s2[1], n_a, a, &ez, MM_F_SPLICE_REV);
 			if (s[0].p->dp_score > s[1].p->dp_score) which = 0, trans_strand = 1;
 			else if (s[0].p->dp_score < s[1].p->dp_score) which = 1, trans_strand = 2;
 			else trans_strand = 3, which = (qlen + s[0].p->dp_score) & 1; // randomly choose a strand, effectively
@@ -476,21 +862,23 @@ mm_reg1_t *mm_align_skeleton(void *km, const mm_mapopt_t *opt, const mm_idx_t *m
 				free(s[0].p);
 			}
 			regs[i].p->trans_strand = trans_strand;
-		} else {
-			mm_align1(km, opt, mi, qlen, qseq0, &regs[i], &r2, a, &ez, opt->flag);
-			if ((opt->flag&MM_F_SPLICE) && !(opt->flag&MM_F_SPLICE_BOTH))
+		} else { // one round of alignment
+			mm_align1(km, opt, mi, qlen, qseq0, &regs[i], &r2, n_a, a, &ez, opt->flag);
+			if (opt->flag&MM_F_SPLICE)
 				regs[i].p->trans_strand = opt->flag&MM_F_SPLICE_FOR? 1 : 2;
 		}
 		if (r2.cnt > 0) regs = mm_insert_reg(&r2, i, &n_regs, regs);
-		if (i > 0 && mm_align1_inv(km, opt, mi, qlen, qseq0, &regs[i-1], &regs[i], &r2, &ez)) {
-			regs = mm_insert_reg(&r2, i, &n_regs, regs);
-			++i; // skip the inserted INV alignment
+		if (i > 0 && regs[i].split_inv) {
+			if (mm_align1_inv(km, opt, mi, qlen, qseq0, &regs[i-1], &regs[i], &r2, &ez)) {
+				regs = mm_insert_reg(&r2, i, &n_regs, regs);
+				++i; // skip the inserted INV alignment
+			}
 		}
 	}
 	*n_regs_ = n_regs;
-	kfree(km, qseq0[0]); kfree(km, qseq0[1]);
+	kfree(km, qseq0[0]);
 	kfree(km, ez.cigar);
-	mm_filter_regs(km, opt, n_regs_, regs);
-	mm_hit_sort_by_dp(km, n_regs_, regs);
+	mm_filter_regs(opt, qlen, n_regs_, regs);
+	mm_hit_sort(km, n_regs_, regs);
 	return regs;
 }

bseq.c

@@ -1,15 +1,38 @@
 #include <zlib.h>
 #include <stdio.h>
 #include <stdlib.h>
-#include <string.h>
 #include <assert.h>
+#define __STDC_LIMIT_MACROS
 #include "bseq.h"
+#include "kvec.h"
 #include "kseq.h"
-KSEQ_INIT(gzFile, gzread)
+KSEQ_INIT2(, gzFile, gzread)
+
+unsigned char seq_comp_table[256] = {
+	  0,   1,	2,	 3,	  4,   5,	6,	 7,	  8,   9,  10,	11,	 12,  13,  14,	15,
+	 16,  17,  18,	19,	 20,  21,  22,	23,	 24,  25,  26,	27,	 28,  29,  30,	31,
+	 32,  33,  34,	35,	 36,  37,  38,	39,	 40,  41,  42,	43,	 44,  45,  46,	47,
+	 48,  49,  50,	51,	 52,  53,  54,	55,	 56,  57,  58,	59,	 60,  61,  62,	63,
+	 64, 'T', 'V', 'G', 'H', 'E', 'F', 'C', 'D', 'I', 'J', 'M', 'L', 'K', 'N', 'O',
+	'P', 'Q', 'Y', 'S', 'A', 'A', 'B', 'W', 'X', 'R', 'Z',	91,	 92,  93,  94,	95,
+	 64, 't', 'v', 'g', 'h', 'e', 'f', 'c', 'd', 'i', 'j', 'm', 'l', 'k', 'n', 'o',
+	'p', 'q', 'y', 's', 'a', 'a', 'b', 'w', 'x', 'r', 'z', 123, 124, 125, 126, 127,
+	128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,
+	144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,
+	160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,
+	176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,
+	192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,
+	208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,
+	224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,
+	240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255
+};
+
+#define CHECK_PAIR_THRES 1000000
 
 struct mm_bseq_file_s {
 	gzFile fp;
 	kseq_t *ks;
+	mm_bseq1_t s;
 };
 
 mm_bseq_file_t *mm_bseq_open(const char *fn)
@@ -31,32 +54,109 @@ void mm_bseq_close(mm_bseq_file_t *fp)
 	free(fp);
 }
 
-mm_bseq1_t *mm_bseq_read(mm_bseq_file_t *fp, int chunk_size, int with_qual, int *n_)
+static inline char *kstrdup(const kstring_t *s)
 {
-	int size = 0, m, n;
-	mm_bseq1_t *seqs;
+	char *t;
+	t = (char*)malloc(s->l + 1);
+	memcpy(t, s->s, s->l + 1);
+	return t;
+}
+
+static inline void kseq2bseq(kseq_t *ks, mm_bseq1_t *s, int with_qual, int with_comment)
+{
+	int i;
+	s->name = kstrdup(&ks->name);
+	s->seq = kstrdup(&ks->seq);
+	for (i = 0; i < (int)ks->seq.l; ++i) // convert U to T
+		if (s->seq[i] == 'u' || s->seq[i] == 'U')
+			--s->seq[i];
+	s->qual = with_qual && ks->qual.l? kstrdup(&ks->qual) : 0;
+	s->comment = with_comment && ks->comment.l? kstrdup(&ks->comment) : 0;
+	s->l_seq = ks->seq.l;
+}
+
+mm_bseq1_t *mm_bseq_read3(mm_bseq_file_t *fp, int chunk_size, int with_qual, int with_comment, int frag_mode, int *n_)
+{
+	int64_t size = 0;
+	kvec_t(mm_bseq1_t) a = {0,0,0};
 	kseq_t *ks = fp->ks;
-	m = n = 0; seqs = 0;
+	*n_ = 0;
+	if (fp->s.seq) {
+		kv_resize(mm_bseq1_t, 0, a, 256);
+		kv_push(mm_bseq1_t, 0, a, fp->s);
+		size = fp->s.l_seq;
+		memset(&fp->s, 0, sizeof(mm_bseq1_t));
+	}
 	while (kseq_read(ks) >= 0) {
 		mm_bseq1_t *s;
 		assert(ks->seq.l <= INT32_MAX);
-		if (n >= m) {
-			m = m? m<<1 : 256;
-			seqs = (mm_bseq1_t*)realloc(seqs, m * sizeof(mm_bseq1_t));
+		if (a.m == 0) kv_resize(mm_bseq1_t, 0, a, 256);
+		kv_pushp(mm_bseq1_t, 0, a, &s);
+		kseq2bseq(ks, s, with_qual, with_comment);
+		size += s->l_seq;
+		if (size >= chunk_size) {
+			if (frag_mode && a.a[a.n-1].l_seq < CHECK_PAIR_THRES) {
+				while (kseq_read(ks) >= 0) {
+					kseq2bseq(ks, &fp->s, with_qual, with_comment);
+					if (mm_qname_same(fp->s.name, a.a[a.n-1].name)) {
+						kv_push(mm_bseq1_t, 0, a, fp->s);
+						memset(&fp->s, 0, sizeof(mm_bseq1_t));
+					} else break;
+				}
+			}
+			break;
+		}
+	}
+	*n_ = a.n;
+	return a.a;
+}
+
+mm_bseq1_t *mm_bseq_read2(mm_bseq_file_t *fp, int chunk_size, int with_qual, int frag_mode, int *n_)
+{
+	return mm_bseq_read3(fp, chunk_size, with_qual, 0, frag_mode, n_);
+}
+
+mm_bseq1_t *mm_bseq_read(mm_bseq_file_t *fp, int chunk_size, int with_qual, int *n_)
+{
+	return mm_bseq_read2(fp, chunk_size, with_qual, 0, n_);
+}
+
+mm_bseq1_t *mm_bseq_read_frag2(int n_fp, mm_bseq_file_t **fp, int chunk_size, int with_qual, int with_comment, int *n_)
+{
+	int i;
+	int64_t size = 0;
+	kvec_t(mm_bseq1_t) a = {0,0,0};
+	*n_ = 0;
+	if (n_fp < 1) return 0;
+	while (1) {
+		int n_read = 0;
+		for (i = 0; i < n_fp; ++i)
+			if (kseq_read(fp[i]->ks) >= 0)
+				++n_read;
+		if (n_read < n_fp) {
+			if (n_read > 0)
+				fprintf(stderr, "[W::%s]\033[1;31m query files have different number of records; extra records skipped.\033[0m\n", __func__);
+			break; // some file reaches the end
+		}
+		if (a.m == 0) kv_resize(mm_bseq1_t, 0, a, 256);
+		for (i = 0; i < n_fp; ++i) {
+			mm_bseq1_t *s;
+			kv_pushp(mm_bseq1_t, 0, a, &s);
+			kseq2bseq(fp[i]->ks, s, with_qual, with_comment);
+			size += s->l_seq;
 		}
-		s = &seqs[n];
-		s->name = strdup(ks->name.s);
-		s->seq = strdup(ks->seq.s);
-		s->qual = with_qual && ks->qual.l? strdup(ks->qual.s) : 0;
-		s->l_seq = ks->seq.l;
-		size += seqs[n++].l_seq;
 		if (size >= chunk_size) break;
 	}
-	*n_ = n;
-	return seqs;
+	*n_ = a.n;
+	return a.a;
+}
+
+mm_bseq1_t *mm_bseq_read_frag(int n_fp, mm_bseq_file_t **fp, int chunk_size, int with_qual, int *n_)
+{
+	return mm_bseq_read_frag2(n_fp, fp, chunk_size, with_qual, 0, n_);
 }
 
 int mm_bseq_eof(mm_bseq_file_t *fp)
 {
-	return ks_eof(fp->ks->f);
+	return (ks_eof(fp->ks->f) && fp->s.seq == 0);
 }

bseq.h

@@ -2,6 +2,7 @@
 #define MM_BSEQ_H
 
 #include <stdint.h>
+#include <string.h>
 
 #ifdef __cplusplus
 extern "C" {
@@ -12,15 +13,49 @@ typedef struct mm_bseq_file_s mm_bseq_file_t;
 
 typedef struct {
 	int l_seq, rid;
-	char *name, *seq, *qual;
+	char *name, *seq, *qual, *comment;
 } mm_bseq1_t;
 
 mm_bseq_file_t *mm_bseq_open(const char *fn);
 void mm_bseq_close(mm_bseq_file_t *fp);
+mm_bseq1_t *mm_bseq_read3(mm_bseq_file_t *fp, int chunk_size, int with_qual, int with_comment, int frag_mode, int *n_);
+mm_bseq1_t *mm_bseq_read2(mm_bseq_file_t *fp, int chunk_size, int with_qual, int frag_mode, int *n_);
 mm_bseq1_t *mm_bseq_read(mm_bseq_file_t *fp, int chunk_size, int with_qual, int *n_);
+mm_bseq1_t *mm_bseq_read_frag2(int n_fp, mm_bseq_file_t **fp, int chunk_size, int with_qual, int with_comment, int *n_);
+mm_bseq1_t *mm_bseq_read_frag(int n_fp, mm_bseq_file_t **fp, int chunk_size, int with_qual, int *n_);
 int mm_bseq_eof(mm_bseq_file_t *fp);
 
 extern unsigned char seq_nt4_table[256];
+extern unsigned char seq_comp_table[256];
+
+static inline int mm_qname_len(const char *s)
+{
+	int l;
+	l = strlen(s);
+	return l >= 3 && s[l-1] >= '0' && s[l-1] <= '9' && s[l-2] == '/'? l - 2 : l;
+}
+
+static inline int mm_qname_same(const char *s1, const char *s2)
+{
+	int l1, l2;
+	l1 = mm_qname_len(s1);
+	l2 = mm_qname_len(s2);
+	return (l1 == l2 && strncmp(s1, s2, l1) == 0);
+}
+
+static inline void mm_revcomp_bseq(mm_bseq1_t *s)
+{
+	int i, t, l = s->l_seq;
+	for (i = 0; i < l>>1; ++i) {
+		t = s->seq[l - i - 1];
+		s->seq[l - i - 1] = seq_comp_table[(uint8_t)s->seq[i]];
+		s->seq[i] = seq_comp_table[t];
+	}
+	if (l&1) s->seq[l>>1] = seq_comp_table[(uint8_t)s->seq[l>>1]];
+	if (s->qual)
+		for (i = 0; i < l>>1; ++i)
+			t = s->qual[l - i - 1], s->qual[l - i - 1] = s->qual[i], s->qual[i] = t;
+}
 
 #ifdef __cplusplus
 }

chain.c

@@ -19,15 +19,15 @@ static inline int ilog2_32(uint32_t v)
 	return (t = v>>8) ? 8 + LogTable256[t] : LogTable256[v];
 }
 
-int mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cnt, int min_sc, int is_cdna, int64_t n, mm128_t *a, uint64_t **_u, void *km)
+mm128_t *mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cnt, int min_sc, int is_cdna, int n_segs, int64_t n, mm128_t *a, int *n_u_, uint64_t **_u, void *km)
 { // TODO: make sure this works when n has more than 32 bits
-	int32_t st = 0, k, *f, *p, *t, *v, n_u, n_v;
-	int64_t i, j;
+	int32_t k, *f, *p, *t, *v, n_u, n_v;
+	int64_t i, j, st = 0;
 	uint64_t *u, *u2, sum_qspan = 0;
 	float avg_qspan;
 	mm128_t *b, *w;
 
-	if (_u) *_u = 0;
+	if (_u) *_u = 0, *n_u_ = 0;
 	f = (int32_t*)kmalloc(km, n * 4);
 	p = (int32_t*)kmalloc(km, n * 4);
 	t = (int32_t*)kmalloc(km, n * 4);
@@ -40,25 +40,31 @@ int mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cn
 	// fill the score and backtrack arrays
 	for (i = 0; i < n; ++i) {
 		uint64_t ri = a[i].x;
+		int64_t max_j = -1;
 		int32_t qi = (int32_t)a[i].y, q_span = a[i].y>>32&0xff; // NB: only 8 bits of span is used!!!
-		int32_t max_f = q_span, max_j = -1, n_skip = 0, min_d, max_f_past = -INT32_MAX;
-		while (st < i && ri - a[st].x > max_dist_x) ++st;
+		int32_t max_f = q_span, n_skip = 0, min_d;
+		int32_t sidi = (a[i].y & MM_SEED_SEG_MASK) >> MM_SEED_SEG_SHIFT;
+		while (st < i && ri > a[st].x + max_dist_x) ++st;
 		for (j = i - 1; j >= st; --j) {
 			int64_t dr = ri - a[j].x;
-			int32_t dq = qi - (int32_t)a[j].y, dd, sc;
-			if (dr == 0 || dq <= 0 || dq > max_dist_y) continue;
+			int32_t dq = qi - (int32_t)a[j].y, dd, sc, log_dd;
+			int32_t sidj = (a[j].y & MM_SEED_SEG_MASK) >> MM_SEED_SEG_SHIFT;
+			if ((sidi == sidj && dr == 0) || dq <= 0) continue; // don't skip if an anchor is used by multiple segments; see below
+			if ((sidi == sidj && dq > max_dist_y) || dq > max_dist_x) continue;
 			dd = dr > dq? dr - dq : dq - dr;
-			if (dd > bw) continue;
-			max_f_past = max_f_past > f[j]? max_f_past : f[j];
+			if (sidi == sidj && dd > bw) continue;
+			if (n_segs > 1 && !is_cdna && sidi == sidj && dr > max_dist_y) continue;
 			min_d = dq < dr? dq : dr;
 			sc = min_d > q_span? q_span : dq < dr? dq : dr;
-			if (is_cdna) {
+			log_dd = dd? ilog2_32(dd) : 0;
+			if (is_cdna || sidi != sidj) {
 				int c_log, c_lin;
 				c_lin = (int)(dd * .01 * avg_qspan);
-				c_log = ilog2_32(dd);
-				if (dr > dq) sc -= c_lin < c_log? c_lin : c_log;
+				c_log = log_dd;
+				if (sidi != sidj && dr == 0) ++sc; // possibly due to overlapping paired ends; give a minor bonus
+				else if (dr > dq || sidi != sidj) sc -= c_lin < c_log? c_lin : c_log;
 				else sc -= c_lin + (c_log>>1);
-			} else sc -= (int)(dd * .01 * avg_qspan) + (ilog2_32(dd)>>1);
+			} else sc -= (int)(dd * .01 * avg_qspan) + (log_dd>>1);
 			sc += f[j];
 			if (sc > max_f) {
 				max_f = sc, max_j = j;
@@ -69,7 +75,8 @@ int mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cn
 			}
 			if (p[j] >= 0) t[p[j]] = i;
 		}
-		f[i] = max_f, p[i] = max_j, v[i] = max_f_past; // v[] keeps the max score in the previous chain
+		f[i] = max_f, p[i] = max_j;
+		v[i] = max_j >= 0 && v[max_j] > max_f? v[max_j] : max_f; // v[] keeps the peak score up to i; f[] is the score ending at i, not always the peak
 	}
 
 	// find the ending positions of chains
@@ -80,14 +87,14 @@ int mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cn
 		if (t[i] == 0 && v[i] >= min_sc)
 			++n_u;
 	if (n_u == 0) {
-		kfree(km, f); kfree(km, p); kfree(km, t); kfree(km, v);
+		kfree(km, a); kfree(km, f); kfree(km, p); kfree(km, t); kfree(km, v);
 		return 0;
 	}
 	u = (uint64_t*)kmalloc(km, n_u * 8);
 	for (i = n_u = 0; i < n; ++i) {
 		if (t[i] == 0 && v[i] >= min_sc) {
 			j = i;
-			while (j >= 0 && f[j] < v[j]) j = p[j]; // find the point that maximizes f[]
+			while (j >= 0 && f[j] < v[j]) j = p[j]; // find the peak that maximizes f[]
 			if (j < 0) j = i; // TODO: this should really be assert(j>=0)
 			u[n_u++] = (uint64_t)f[j] << 32 | j;
 		}
@@ -115,9 +122,9 @@ int mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cn
 		}
 		if (k0 == k) n_v = n_v0; // no new chain added, reset
 	}
-	n_u = k, *_u = u; // NB: note that u[] may not be sorted by score here
+	*n_u_ = n_u = k, *_u = u; // NB: note that u[] may not be sorted by score here
 
-	// free
+	// free temporary arrays
 	kfree(km, f); kfree(km, p); kfree(km, t);
 
 	// write the result to b[]
@@ -144,6 +151,7 @@ int mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cn
 		k += n;
 	}
 	memcpy(u, u2, n_u * 8);
-	kfree(km, b); kfree(km, w); kfree(km, u2);
-	return n_u;
+	memcpy(b, a, k * sizeof(mm128_t)); // write _a_ to _b_ and deallocate _a_ because _a_ is oversized, sometimes a lot
+	kfree(km, a); kfree(km, w); kfree(km, u2);
+	return b;
 }

cookbook.md

@@ -0,0 +1,243 @@
+## Table of Contents
+
+- [Introduction & Installation](#intro)
+- [Mapping Genomic Reads](#map-reads)
+  * [Mapping long reads](#map-pb)
+  * [Mapping Illumina paired-end reads](#map-sr)
+  * [Evaluating mapping accuracy with simulated reads (for developers)](#mapeval)
+- [Mapping Long RNA-seq Reads](#map-rna)
+  * [Mapping Nanopore 2D cDNA reads](#map-ont-cdna-2d)
+  * [Mapping Nanopore direct-RNA reads](#map-direct-rna)
+  * [Mapping PacBio Iso-seq reads](#map-iso-seq)
+- [Full-Genome Alignment](#genome-aln)
+  * [Intra-species assembly alignment](#asm-to-ref)
+  * [Cross-species full-genome alignment](#x-species)
+  * [Eyeballing alignment](#view-aln)
+  * [Calling variants from assembly-to-reference alignment](#asm-var)
+  * [Constructing self-homology map](#hom-map)
+  * [Lift Over (for developers)](#liftover)
+- [Read Overlap](#read-overlap)
+  * [Long-read overlap](#long-read-overlap)
+  * [Evaluating overlap sensitivity (for developers)](#ov-eval)
+
+## <a name="intro"></a>Introduction & Installation
+
+This cookbook walks you through a variety of applications of minimap2 and its
+companion script `paftools.js`. All data here are freely available from the
+minimap2 release page at version tag [v2.10][v2.10]. Some examples only work
+with v2.10 or later.
+
+To acquire the data used in this cookbook and to install minimap2 and paftools,
+please follow the command lines below:
+```sh
+# install minimap2 executables
+curl -L https://github.com/lh3/minimap2/releases/download/v2.13/minimap2-2.13_x64-linux.tar.bz2 | tar jxf -
+cp minimap2-2.13_x64-linux/{minimap2,k8,paftools.js} .  # copy executables
+export PATH="$PATH:"`pwd`                               # put the current directory on PATH
+# download example datasets
+curl -L https://github.com/lh3/minimap2/releases/download/v2.10/cookbook-data.tgz | tar zxf -
+```
+
+## <a name="map-reads"></a>Mapping Genomic Reads
+
+### <a name="map-pb"></a>Mapping long reads
+```sh
+minimap2 -ax map-pb -t4 ecoli_ref.fa ecoli_p6_25x_canu.fa > mapped.sam
+```
+Alternatively, you can create a minimap2 index first and then map:
+```sh
+minimap2 -x map-pb -d ecoli-pb.mmi ecoli_ref.fa                      # create an index
+minimap2 -ax map-pb ecoli-pb.mmi ecoli_p6_25x_canu.fa > mapped.sam
+```
+This will save you a couple of minutes when you map against the human genome.
+**HOWEVER**, key algorithm parameters such as the k-mer length and window
+size can't be changed after indexing. Minimap2 will give you a warning if
+parameters used in a pre-built index doesn't match parameters on the command
+line. **Please always make sure you are using an intended pre-built index.**
+
+### <a name="map-sr"></a>Mapping Illumina paired-end reads:
+```sh
+minimap2 -ax sr -t4 ecoli_ref.fa ecoli_mason_1.fq ecoli_mason_2.fq > mapped-sr.sam
+```
+
+### <a name="mapeval"></a>Evaluating mapping accuracy with simulated reads (for developers)
+```sh
+minimap2 -ax sr ecoli_ref.fa ecoli_mason_1.fq ecoli_mason_2.fq | paftools.js mapeval -
+```
+The output is:
+```
+Q       60      19712   0       0.000000000     19712
+Q       0       282     219     0.010953286     19994
+U       6
+```
+where a `U`-line gives the number of unmapped reads (for SAM input only); a
+`Q`-line gives:
+
+1. Mapping quality (mapQ) threshold
+2. Number of mapped reads between this threshold and the previous mapQ threshold.
+3. Number of wrong mappings in the same mapQ interval
+4. Accumulative mapping error rate
+5. Accumulative number of mappings
+
+For `paftools.js mapeval` to work, you need to encode the true read positions
+in read names in the right format. For [PBSIM][pbsim] and [mason2][mason2], we
+provide scripts to generate the right format. Simulated reads in this cookbook
+were created with the following command lines:
+```sh
+# in PBSIM source code directory:
+src/pbsim ../ecoli_ref.fa --depth 1 --sample-fastq sample/sample.fastq
+paftools.js pbsim2fq ../ecoli_ref.fa.fai sd_0001.maf > ../ecoli_pbsim.fa
+
+# mason2 simulation
+mason_simulator --illumina-prob-mismatch-scale 2.5 -ir ecoli_ref.fa -n 10000 -o tmp-l.fq -or tmp-r.fq -oa tmp.sam
+paftools.js mason2fq tmp.sam | seqtk seq -1 > ecoli_mason_1.fq
+paftools.js mason2fq tmp.sam | seqtk seq -2 > ecoli_mason_2.fq
+```
+
+
+
+## <a name="map-rna"></a>Mapping Long RNA-seq Reads
+
+### <a name="map-ont-cdna-2d"></a>Mapping Nanopore 2D cDNA reads
+```sh
+minimap2 -ax splice SIRV_E2.fa SIRV_ont-cdna.fa > aln.sam
+```
+You can compare the alignment to the true annotations with:
+```sh
+paftools.js junceval SIRV_E2C.gtf aln.sam
+```
+It gives the percentage of introns found in the annotation. For SIRV data, it
+is possible to achieve higher junction accuracy with
+```sh
+minimap2 -ax splice --splice-flank=no SIRV_E2.fa SIRV_ont-cdna.fa | paftools.js junceval SIRV_E2C.gtf
+```
+This is because minimap2 models one additional evolutionarily conserved base
+around a canonical junction, but SIRV doesn't honor this signal. Option
+`--splice-flank=no` asks minimap2 no to model this additional base.
+
+In the output a tag `ts:A:+` indicates that the read strand is the same as the
+transcript strand; `ts:A:-` indicates the read strand is opposite to the
+transcript strand. This tag is inferred from the GT-AG signal and is thus only
+available to spliced reads.
+
+### <a name="map-direct-rna"></a>Mapping Nanopore direct-RNA reads
+```sh
+minimap2 -ax splice -k14 -uf SIRV_E2.fa SIRV_ont-drna.fa > aln.sam
+```
+Direct-RNA reads are noisier, so we use a shorter k-mer for improved
+sensitivity. Here, option `-uf` forces minimap2 to map reads to the forward
+transcript strand only because direct-RNA reads are stranded. Again, applying
+`--splice-flank=no` helps junction accuracy for SIRV data.
+
+### <a name="map-iso-seq"></a>Mapping PacBio Iso-seq reads
+```sh
+minimap2 -ax splice -uf -C5 SIRV_E2.fa SIRV_iso-seq.fq > aln.sam
+```
+Option `-C5` reduces the penalty on non-canonical splicing sites. It helps
+to align such sites correctly for data with low error rate such as Iso-seq
+reads and traditional cDNAs. On this example, minimap2 makes one junction
+error. Applying `--splice-flank=no` fixes this alignment error.
+
+Note that the command line above is optimized for the final Iso-seq reads.
+PacBio's Iso-seq pipeline produces intermediate sequences at varying quality.
+For example, some intermediate reads are not stranded. For these reads, option
+`-uf` will lead to more errors. Please revise the minimap2 command line
+accordingly.
+
+
+
+## <a name="genome-aln"></a>Full-Genome Alignment
+
+### <a name="asm-to-ref"></a>Intra-species assembly alignment
+```sh
+# option "--cs" is recommended as paftools.js may need it
+minimap2 -cx asm5 --cs ecoli_ref.fa ecoli_canu.fa > ecoli_canu.paf
+```
+Here `ecoli_canu.fa` is the Canu assembly of `ecoli_p6_25x_canu.fa`. This
+command line outputs alignments in the [PAF format][paf]. Use `-a` instead of
+`-c` to get output in the SAM format.
+
+### <a name="x-species"></a>Cross-species full-genome alignment
+```sh
+minimap2 -cx asm20 --cs ecoli_ref.fa ecoli_O104:H4.fa > ecoli_O104:H4.paf
+sort -k6,6 -k8,8n ecoli_O104:H4.paf | paftools.js call -f ecoli_ref.fa -L10000 -l1000 - > out.vcf
+```
+Minimap2 has three presets for full-genome alignment: "asm5" for sequence
+divergence below 1%, "asm10" for divergence around a couple of percent and
+"asm20" for divergence not more than 10%. In theory, with the right setting,
+minimap2 should work for sequence pairs with sequence divergence up to ~15%,
+but this has not been carefully evaluated.
+
+### <a name="view-aln"></a>Eyeballing alignment
+```sh
+# option "--cs" required; minimap2-r741 or higher required for the "asm20" preset
+minimap2 -cx asm20 --cs ecoli_ref.fa ecoli_O104:H4.fa | paftools.js view - | less -S
+```
+This prints the alignment in a BLAST-like format.
+
+### <a name="asm-var"></a>Calling variants from assembly-to-reference alignment
+```sh
+# don't forget the "--cs" option; otherwise it doesn't work
+minimap2 -cx asm5 --cs ecoli_ref.fa ecoli_canu.fa \
+  | sort -k6,6 -k8,8n \
+  | paftools.js call -f ecoli_ref.fa - > out.vcf
+```
+Without option `-f`, `paftools.js call` outputs in a custom format. In this
+format, lines starting with `R` give the regions covered by one contig only.
+This information is not available in the VCF output.
+
+### <a name="hom-map"></a>Constructing self-homology map
+```sh
+minimap2 -DP -k19 -w19 -m200 ecoli_ref.fa ecoli_ref.fa > out.paf
+```
+Option `-D` asks minimap2 to ignore anchors from perfect self match and `-P`
+outputs all chains. For large nomes, we don't recommend to perform base-level
+alignment (with `-c`, `-a` or `--cs`) when `-P` is applied. This is because
+base-alignment is slow and occasionally gives wrong alignments close to the
+diagonal of a dotter plot. For E. coli, though, base-alignment is still fast.
+
+### <a name="liftover"></a>Lift over (for developers)
+```sh
+minimap2 -cx asm5 --cs ecoli_ref.fa ecoli_canu.fa > ecoli_canu.paf
+echo -e 'tig00000001\t200000\t300000' | paftools.js liftover ecoli_canu.paf -
+```
+This lifts over a region on query sequences to one or multiple regions on
+reference sequences. Note that this paftools.js command may not be efficient
+enough to lift millions of regions.
+
+
+
+## <a name="read-overlap"></a>Read Overlap
+
+### <a name="long-read-overlap"></a>Long read overlap
+```sh
+# For pacbio reads:
+minimap2 -x ava-pb ecoli_p6_25x_canu.fa ecoli_p6_25x_canu.fa > overlap.paf
+# For Nanopore reads (ava-ont also works with PacBio but not as good):
+minimap2 -x ava-ont -r 10000 ecoli_p6_25x_canu.fa ecoli_p6_25x_canu.fa > overlap.paf
+# If you have miniasm installed:
+miniasm -f ecoli_p6_25x_canu.fa overlap.paf > asm.gfa
+```
+Here we explicitly applied `-r 10000`. We are considering to set this as the
+default for the `ava-ont` mode as this seems to improve the contiguity for
+nanopore read assembly (Loman, personal communication).
+
+*Minimap2 doesn't work well with short-read overlap.*
+
+### <a name="ov-eval"></a>Evaluating overlap sensitivity (for developers)
+
+```sh
+# read to reference mapping
+minimap2 -cx map-pb ecoli_ref.fa ecoli_p6_25x_canu.fa > to-ref.paf
+# evaluate overlap sensitivity
+sort -k6,6 -k8,8n to-ref.paf | paftools.js ov-eval - overlap.paf
+```
+You can see that for PacBio reads, minimap2 achieves higher overlap sensitivity
+with `-x ava-pb` (99% vs 93% with `-x ava-ont`).
+
+
+
+[pbsim]: https://github.com/pfaucon/PBSIM-PacBio-Simulator
+[mason2]: https://github.com/seqan/seqan/tree/master/apps/mason2
+[paf]: https://github.com/lh3/miniasm/blob/master/PAF.md
+[v2.10]: https://github.com/lh3/minimap2/releases/tag/v2.10

esterr.c

@@ -0,0 +1,64 @@
+#include <math.h>
+#include <stdio.h>
+#include <stdlib.h>
+#include <assert.h>
+#include "mmpriv.h"
+
+static inline int32_t get_for_qpos(int32_t qlen, const mm128_t *a)
+{
+	int32_t x = (int32_t)a->y;
+	int32_t q_span = a->y>>32 & 0xff;
+	if (a->x>>63)
+		x = qlen - 1 - (x + 1 - q_span); // revert the position to the forward strand of query
+	return x;
+}
+
+static int get_mini_idx(int qlen, const mm128_t *a, int32_t n, const uint64_t *mini_pos)
+{
+	int32_t x, L = 0, R = n - 1;
+	x = get_for_qpos(qlen, a);
+	while (L <= R) { // binary search
+		int32_t m = ((uint64_t)L + R) >> 1;
+		int32_t y = (int32_t)mini_pos[m];
+		if (y < x) L = m + 1;
+		else if (y > x) R = m - 1;
+		else return m;
+	}
+	return -1;
+}
+
+void mm_est_err(const mm_idx_t *mi, int qlen, int n_regs, mm_reg1_t *regs, const mm128_t *a, int32_t n, const uint64_t *mini_pos)
+{
+	int i;
+	uint64_t sum_k = 0;
+	float avg_k;
+
+	if (n == 0) return;
+	for (i = 0; i < n; ++i)
+		sum_k += mini_pos[i] >> 32 & 0xff;
+	avg_k = (float)sum_k / n;
+
+	for (i = 0; i < n_regs; ++i) {
+		mm_reg1_t *r = &regs[i];
+		int32_t st, en, j, k, n_match, n_tot, l_ref;
+		r->div = -1.0f;
+		if (r->cnt == 0) continue;
+		st = en = get_mini_idx(qlen, r->rev? &a[r->as + r->cnt - 1] : &a[r->as], n, mini_pos);
+		if (st < 0) {
+			if (mm_verbose >= 2)
+				fprintf(stderr, "[WARNING] logic inconsistency in mm_est_err(). Please contact the developer.\n");
+			continue;
+		}
+		l_ref = mi->seq[r->rid].len;
+		for (k = 1, j = st + 1, n_match = 1; j < n && k < r->cnt; ++j) {
+			int32_t x;
+			x = get_for_qpos(qlen, r->rev? &a[r->as + r->cnt - 1 - k] : &a[r->as + k]);
+			if (x == (int32_t)mini_pos[j])
+				++k, en = j, ++n_match;
+		}
+		n_tot = en - st + 1;
+		if (r->qs > avg_k && r->rs > avg_k) ++n_tot;
+		if (qlen - r->qs > avg_k && l_ref - r->re > avg_k) ++n_tot;
+		r->div = logf((float)n_tot / n_match) / avg_k;
+	}
+}

example.c

@@ -11,51 +11,51 @@ KSEQ_INIT(gzFile, gzread)
 
 int main(int argc, char *argv[])
 {
+	mm_idxopt_t iopt;
+	mm_mapopt_t mopt;
+	int n_threads = 3;
+
 	mm_verbose = 2; // disable message output to stderr
+	mm_set_opt(0, &iopt, &mopt);
+	mopt.flag |= MM_F_CIGAR; // perform alignment
 
 	if (argc < 3) {
 		fprintf(stderr, "Usage: minimap2-lite <target.fa> <query.fa>\n");
 		return 1;
 	}
-	
+
 	// open query file for reading; you may use your favorite FASTA/Q parser
 	gzFile f = gzopen(argv[2], "r");
 	assert(f);
 	kseq_t *ks = kseq_init(f);
 
-	// create index for target; we are creating one index for all target sequence
-	int n_threads = 4, w = 10, k = 15, is_hpc = 0;
-	mm_idx_t *mi = mm_idx_build(argv[1], w, k, is_hpc, n_threads);
-	assert(mi);
-
-	// mapping
-	mm_mapopt_t opt;
-	mm_mapopt_init(&opt); // initialize mapping parameters
-	mm_mapopt_update(&opt, mi); // this sets the maximum minimizer occurrence; TODO: set a better default in mm_mapopt_init()!
-	opt.flag |= MM_F_CIGAR; // perform alignment
-	mm_tbuf_t *tbuf = mm_tbuf_init(); // thread buffer; for multi-threading, allocate one tbuf for each thread
-	while (kseq_read(ks) >= 0) { // each kseq_read() call reads one query sequence
-		const mm_reg1_t *reg;
-		int j, i, n_reg;
-		// get all hits for the query
-		reg = mm_map(mi, ks->seq.l, ks->seq.s, &n_reg, tbuf, &opt, 0);
-		// traverse hits and print them out
-		for (j = 0; j < n_reg; ++j) {
-			const mm_reg1_t *r = &reg[j];
-			assert(r->p); // with MM_F_CIGAR, this should not be NULL
-			printf("%s\t%d\t%d\t%d\t%c\t", ks->name.s, ks->seq.l, r->qs, r->qe, "+-"[r->rev]);
-			printf("%s\t%d\t%d\t%d\t%d\t%d\t%d\tcg:Z:", mi->seq[r->rid].name, mi->seq[r->rid].len, r->rs, r->re,
-				   r->p->blen - r->p->n_ambi - r->p->n_diff, r->p->blen, r->mapq);
-			for (i = 0; i < r->p->n_cigar; ++i) // IMPORTANT: this gives the CIGAR in the aligned regions. NO soft/hard clippings!
-				printf("%d%c", r->p->cigar[i]>>4, "MIDSHN"[r->p->cigar[i]&0xf]);
-			putchar('\n');
+	// open index reader
+	mm_idx_reader_t *r = mm_idx_reader_open(argv[1], &iopt, 0);
+	mm_idx_t *mi;
+	while ((mi = mm_idx_reader_read(r, n_threads)) != 0) { // traverse each part of the index
+		mm_mapopt_update(&mopt, mi); // this sets the maximum minimizer occurrence; TODO: set a better default in mm_mapopt_init()!
+		mm_tbuf_t *tbuf = mm_tbuf_init(); // thread buffer; for multi-threading, allocate one tbuf for each thread
+		while (kseq_read(ks) >= 0) { // each kseq_read() call reads one query sequence
+			mm_reg1_t *reg;
+			int j, i, n_reg;
+			reg = mm_map(mi, ks->seq.l, ks->seq.s, &n_reg, tbuf, &mopt, 0); // get all hits for the query
+			for (j = 0; j < n_reg; ++j) { // traverse hits and print them out
+				mm_reg1_t *r = &reg[j];
+				assert(r->p); // with MM_F_CIGAR, this should not be NULL
+				printf("%s\t%d\t%d\t%d\t%c\t", ks->name.s, ks->seq.l, r->qs, r->qe, "+-"[r->rev]);
+				printf("%s\t%d\t%d\t%d\t%d\t%d\t%d\tcg:Z:", mi->seq[r->rid].name, mi->seq[r->rid].len, r->rs, r->re, r->mlen, r->blen, r->mapq);
+				for (i = 0; i < r->p->n_cigar; ++i) // IMPORTANT: this gives the CIGAR in the aligned regions. NO soft/hard clippings!
+					printf("%d%c", r->p->cigar[i]>>4, "MIDSHN"[r->p->cigar[i]&0xf]);
+				putchar('\n');
+				free(r->p);
+			}
+			free(reg);
 		}
+		mm_tbuf_destroy(tbuf);
+		mm_idx_destroy(mi);
 	}
-	mm_tbuf_destroy(tbuf);
-
-	// deallocate index and close the query file
-	mm_idx_destroy(mi);
-	kseq_destroy(ks);
+	mm_idx_reader_close(r); // close the index reader
+	kseq_destroy(ks); // close the query file
 	gzclose(f);
 	return 0;
 }

format.c

@@ -43,6 +43,13 @@ static void mm_sprintf_lite(kstring_t *s, const char *fmt, ...)
 				if (c < 0) buf[l++] = '-';
 				str_enlarge(s, l);
 				for (i = l - 1; i >= 0; --i) s->s[s->l++] = buf[i];
+			} else if (*p == 'u') {
+				int i, l = 0;
+				uint32_t x;
+				x = va_arg(ap, uint32_t);
+				do { buf[l++] = x%10 + '0'; x /= 10; } while (x > 0);
+				str_enlarge(s, l);
+				for (i = l - 1; i >= 0; --i) s->s[s->l++] = buf[i];
 			} else if (*p == 's') {
 				char *r = va_arg(ap, char*);
 				str_copy(s, r, r + strlen(r));
@@ -105,10 +112,15 @@ err_set_rg:
 	free(rg_line);
 }
 
-void mm_write_sam_hdr_no_SQ(const char *rg, const char *ver, int argc, char *argv[])
+void mm_write_sam_hdr(const mm_idx_t *idx, const char *rg, const char *ver, int argc, char *argv[])
 {
 	kstring_t str = {0,0,0};
-	sam_write_rg_line(&str, rg);
+	if (idx) {
+		uint32_t i;
+		for (i = 0; i < idx->n_seq; ++i)
+			mm_sprintf_lite(&str, "@SQ\tSN:%s\tLN:%d\n", idx->seq[i].name, idx->seq[i].len);
+	}
+	if (rg) sam_write_rg_line(&str, rg);
 	mm_sprintf_lite(&str, "@PG\tID:minimap2\tPN:minimap2");
 	if (ver) mm_sprintf_lite(&str, "\tVN:%s", ver);
 	if (argc > 1) {
@@ -117,19 +129,96 @@ void mm_write_sam_hdr_no_SQ(const char *rg, const char *ver, int argc, char *arg
 		for (i = 1; i < argc; ++i)
 			mm_sprintf_lite(&str, " %s", argv[i]);
 	}
-	mm_sprintf_lite(&str, "\n");
-	fputs(str.s, stdout);
+	mm_err_puts(str.s);
 	free(str.s);
 }
 
-static void write_cs(void *km, kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const mm_reg1_t *r)
+static void write_cs_core(kstring_t *s, const uint8_t *tseq, const uint8_t *qseq, const mm_reg1_t *r, char *tmp, int no_iden, int write_tag)
+{
+	int i, q_off, t_off;
+	if (write_tag) mm_sprintf_lite(s, "\tcs:Z:");
+	for (i = q_off = t_off = 0; i < (int)r->p->n_cigar; ++i) {
+		int j, op = r->p->cigar[i]&0xf, len = r->p->cigar[i]>>4;
+		assert(op >= 0 && op <= 3);
+		if (op == 0) { // match
+			int l_tmp = 0;
+			for (j = 0; j < len; ++j) {
+				if (qseq[q_off + j] != tseq[t_off + j]) {
+					if (l_tmp > 0) {
+						if (!no_iden) {
+							tmp[l_tmp] = 0;
+							mm_sprintf_lite(s, "=%s", tmp);
+						} else mm_sprintf_lite(s, ":%d", l_tmp);
+						l_tmp = 0;
+					}
+					mm_sprintf_lite(s, "*%c%c", "acgtn"[tseq[t_off + j]], "acgtn"[qseq[q_off + j]]);
+				} else tmp[l_tmp++] = "ACGTN"[qseq[q_off + j]];
+			}
+			if (l_tmp > 0) {
+				if (!no_iden) {
+					tmp[l_tmp] = 0;
+					mm_sprintf_lite(s, "=%s", tmp);
+				} else mm_sprintf_lite(s, ":%d", l_tmp);
+			}
+			q_off += len, t_off += len;
+		} else if (op == 1) { // insertion to ref
+			for (j = 0, tmp[len] = 0; j < len; ++j)
+				tmp[j] = "acgtn"[qseq[q_off + j]];
+			mm_sprintf_lite(s, "+%s", tmp);
+			q_off += len;
+		} else if (op == 2) { // deletion from ref
+			for (j = 0, tmp[len] = 0; j < len; ++j)
+				tmp[j] = "acgtn"[tseq[t_off + j]];
+			mm_sprintf_lite(s, "-%s", tmp);
+			t_off += len;
+		} else { // intron
+			assert(len >= 2);
+			mm_sprintf_lite(s, "~%c%c%d%c%c", "acgtn"[tseq[t_off]], "acgtn"[tseq[t_off+1]],
+				len, "acgtn"[tseq[t_off+len-2]], "acgtn"[tseq[t_off+len-1]]);
+			t_off += len;
+		}
+	}
+	assert(t_off == r->re - r->rs && q_off == r->qe - r->qs);
+}
+
+static void write_MD_core(kstring_t *s, const uint8_t *tseq, const uint8_t *qseq, const mm_reg1_t *r, char *tmp, int write_tag)
+{
+	int i, q_off, t_off, l_MD = 0;
+	if (write_tag) mm_sprintf_lite(s, "\tMD:Z:");
+	for (i = q_off = t_off = 0; i < (int)r->p->n_cigar; ++i) {
+		int j, op = r->p->cigar[i]&0xf, len = r->p->cigar[i]>>4;
+		assert(op >= 0 && op <= 3);
+		if (op == 0) { // match
+			for (j = 0; j < len; ++j) {
+				if (qseq[q_off + j] != tseq[t_off + j]) {
+					mm_sprintf_lite(s, "%d%c", l_MD, "ACGTN"[tseq[t_off + j]]);
+					l_MD = 0;
+				} else ++l_MD;
+			}
+			q_off += len, t_off += len;
+		} else if (op == 1) { // insertion to ref
+			q_off += len;
+		} else if (op == 2) { // deletion from ref
+			for (j = 0, tmp[len] = 0; j < len; ++j)
+				tmp[j] = "ACGTN"[tseq[t_off + j]];
+			mm_sprintf_lite(s, "%d^%s", l_MD, tmp);
+			l_MD = 0;
+			t_off += len;
+		} else if (op == 3) { // reference skip
+			t_off += len;
+		}
+	}
+	if (l_MD > 0) mm_sprintf_lite(s, "%d", l_MD);
+	assert(t_off == r->re - r->rs && q_off == r->qe - r->qs);
+}
+
+static void write_cs_or_MD(void *km, kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const mm_reg1_t *r, int no_iden, int is_MD, int write_tag)
 {
 	extern unsigned char seq_nt4_table[256];
-	int i, q_off, t_off;
+	int i;
 	uint8_t *qseq, *tseq;
 	char *tmp;
 	if (r->p == 0) return;
-	mm_sprintf_lite(s, "\tcs:Z:");
 	qseq = (uint8_t*)kmalloc(km, r->qe - r->qs);
 	tseq = (uint8_t*)kmalloc(km, r->re - r->rs);
 	tmp = (char*)kmalloc(km, r->re - r->rs > r->qe - r->qs? r->re - r->rs + 1 : r->qe - r->qs + 1);
@@ -143,133 +232,232 @@ static void write_cs(void *km, kstring_t *s, const mm_idx_t *mi, const mm_bseq1_
 			qseq[r->qe - i - 1] = c >= 4? 4 : 3 - c;
 		}
 	}
-	for (i = q_off = t_off = 0; i < r->p->n_cigar; ++i) {
-		int j, op = r->p->cigar[i]&0xf, len = r->p->cigar[i]>>4;
-		assert(op >= 0 && op <= 2);
-		if (op == 0) {
-			int l_tmp = 0;
-			for (j = 0; j < len; ++j) {
-				if (qseq[q_off + j] != tseq[t_off + j]) {
-					if (l_tmp > 0) {
-						tmp[l_tmp] = 0;
-						mm_sprintf_lite(s, "=%s", tmp);
-						l_tmp = 0;
-					}
-					mm_sprintf_lite(s, "*%c%c", "acgtn"[tseq[t_off + j]], "acgtn"[qseq[q_off + j]]);
-				} else tmp[l_tmp++] = "ACGTN"[qseq[q_off + j]];
-			}
-			if (l_tmp > 0) {
-				tmp[l_tmp] = 0;
-				mm_sprintf_lite(s, "=%s", tmp);
-			}
-			q_off += len, t_off += len;
-		} else if (op == 1) {
-			for (j = 0, tmp[len] = 0; j < len; ++j)
-				tmp[j] = "acgtn"[qseq[q_off + j]];
-			mm_sprintf_lite(s, "+%s", tmp);
-			q_off += len;
-		} else if (op == 2) {
-			for (j = 0, tmp[len] = 0; j < len; ++j)
-				tmp[j] = "acgtn"[tseq[t_off + j]];
-			mm_sprintf_lite(s, "-%s", tmp);
-			t_off += len;
-		}
-	}
-	assert(t_off == r->re - r->rs && q_off == r->qe - r->qs);
+	if (is_MD) write_MD_core(s, tseq, qseq, r, tmp, write_tag);
+	else write_cs_core(s, tseq, qseq, r, tmp, no_iden, write_tag);
 	kfree(km, qseq); kfree(km, tseq); kfree(km, tmp);
 }
 
+int mm_gen_cs_or_MD(void *km, char **buf, int *max_len, const mm_idx_t *mi, const mm_reg1_t *r, const char *seq, int is_MD, int no_iden)
+{
+	mm_bseq1_t t;
+	kstring_t str;
+	str.s = *buf, str.l = 0, str.m = *max_len;
+	t.l_seq = strlen(seq);
+	t.seq = (char*)seq;
+	write_cs_or_MD(km, &str, mi, &t, r, no_iden, is_MD, 0);
+	*max_len = str.m;
+	*buf = str.s;
+	return str.l;
+}
+
+int mm_gen_cs(void *km, char **buf, int *max_len, const mm_idx_t *mi, const mm_reg1_t *r, const char *seq, int no_iden)
+{
+	return mm_gen_cs_or_MD(km, buf, max_len, mi, r, seq, 0, no_iden);
+}
+
+int mm_gen_MD(void *km, char **buf, int *max_len, const mm_idx_t *mi, const mm_reg1_t *r, const char *seq)
+{
+	return mm_gen_cs_or_MD(km, buf, max_len, mi, r, seq, 1, 0);
+}
+
 static inline void write_tags(kstring_t *s, const mm_reg1_t *r)
 {
-	int type = r->inv? 'I' : r->id == r->parent? 'P' : 'S';
-	mm_sprintf_lite(s, "\ttp:A:%c\tcm:i:%d\ts1:i:%d", type, r->cnt, r->score);
-	if (r->parent == r->id) mm_sprintf_lite(s, "\ts2:i:%d", r->subsc);
-	if (r->split) mm_sprintf_lite(s, "\tzd:i:%d", r->split);
+	int type;
+	if (r->id == r->parent) type = r->inv? 'I' : 'P';
+	else type = r->inv? 'i' : 'S';
 	if (r->p) {
-		mm_sprintf_lite(s, "\tNM:i:%d\tms:i:%d\tAS:i:%d\tnn:i:%d", r->p->n_diff, r->p->dp_max, r->p->dp_score, r->p->n_ambi);
+		mm_sprintf_lite(s, "\tNM:i:%d\tms:i:%d\tAS:i:%d\tnn:i:%d", r->blen - r->mlen + r->p->n_ambi, r->p->dp_max, r->p->dp_score, r->p->n_ambi);
 		if (r->p->trans_strand == 1 || r->p->trans_strand == 2)
 			mm_sprintf_lite(s, "\tts:A:%c", "?+-?"[r->p->trans_strand]);
 	}
+	mm_sprintf_lite(s, "\ttp:A:%c\tcm:i:%d\ts1:i:%d", type, r->cnt, r->score);
+	if (r->parent == r->id) mm_sprintf_lite(s, "\ts2:i:%d", r->subsc);
+	if (r->div >= 0.0f && r->div <= 1.0f) {
+		char buf[8];
+		if (r->div == 0.0f) buf[0] = '0', buf[1] = 0;
+		else sprintf(buf, "%.4f", r->div);
+		mm_sprintf_lite(s, "\tdv:f:%s", buf);
+	}
+	if (r->split) mm_sprintf_lite(s, "\tzd:i:%d", r->split);
 }
 
 void mm_write_paf(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const mm_reg1_t *r, void *km, int opt_flag)
 {
 	s->l = 0;
+	if (r == 0) {
+		mm_sprintf_lite(s, "%s\t%d", t->name, t->l_seq);
+		return;
+	}
 	mm_sprintf_lite(s, "%s\t%d\t%d\t%d\t%c\t", t->name, t->l_seq, r->qs, r->qe, "+-"[r->rev]);
 	if (mi->seq[r->rid].name) mm_sprintf_lite(s, "%s", mi->seq[r->rid].name);
 	else mm_sprintf_lite(s, "%d", r->rid);
 	mm_sprintf_lite(s, "\t%d\t%d\t%d", mi->seq[r->rid].len, r->rs, r->re);
-	if (r->p) mm_sprintf_lite(s, "\t%d\t%d", r->p->blen - r->p->n_ambi - r->p->n_diff, r->p->blen);
-	else mm_sprintf_lite(s, "\t%d\t%d", r->fuzzy_mlen, r->fuzzy_blen);
+	mm_sprintf_lite(s, "\t%d\t%d", r->mlen, r->blen);
 	mm_sprintf_lite(s, "\t%d", r->mapq);
 	write_tags(s, r);
 	if (r->p && (opt_flag & MM_F_OUT_CG)) {
 		uint32_t k;
 		mm_sprintf_lite(s, "\tcg:Z:");
 		for (k = 0; k < r->p->n_cigar; ++k)
-			mm_sprintf_lite(s, "%d%c", r->p->cigar[k]>>4, "MIDN"[r->p->cigar[k]&0xf]);
+			mm_sprintf_lite(s, "%d%c", r->p->cigar[k]>>4, "MIDNSHP=XB"[r->p->cigar[k]&0xf]);
 	}
-	if (r->p && (opt_flag & MM_F_OUT_CS))
-		write_cs(km, s, mi, t, r);
-}
-
-static char comp_tab[] = {
-	  0,   1,	2,	 3,	  4,   5,	6,	 7,	  8,   9,  10,	11,	 12,  13,  14,	15,
-	 16,  17,  18,	19,	 20,  21,  22,	23,	 24,  25,  26,	27,	 28,  29,  30,	31,
-	 32,  33,  34,	35,	 36,  37,  38,	39,	 40,  41,  42,	43,	 44,  45,  46,	47,
-	 48,  49,  50,	51,	 52,  53,  54,	55,	 56,  57,  58,	59,	 60,  61,  62,	63,
-	 64, 'T', 'V', 'G', 'H', 'E', 'F', 'C', 'D', 'I', 'J', 'M', 'L', 'K', 'N', 'O',
-	'P', 'Q', 'Y', 'S', 'A', 'A', 'B', 'W', 'X', 'R', 'Z',	91,	 92,  93,  94,	95,
-	 64, 't', 'v', 'g', 'h', 'e', 'f', 'c', 'd', 'i', 'j', 'm', 'l', 'k', 'n', 'o',
-	'p', 'q', 'y', 's', 'a', 'a', 'b', 'w', 'x', 'r', 'z', 123, 124, 125, 126, 127
-};
-
-void mm_write_sam_SQ(const mm_idx_t *idx)
-{
-	uint32_t i;
-	for (i = 0; i < idx->n_seq; ++i)
-		printf("@SQ\tSN:%s\tLN:%d\n", idx->seq[i].name, idx->seq[i].len);
+	if (r->p && (opt_flag & (MM_F_OUT_CS|MM_F_OUT_MD)))
+		write_cs_or_MD(km, s, mi, t, r, !(opt_flag&MM_F_OUT_CS_LONG), opt_flag&MM_F_OUT_MD, 1);
+	if ((opt_flag & MM_F_COPY_COMMENT) && t->comment)
+		mm_sprintf_lite(s, "\t%s", t->comment);
 }
 
 static void sam_write_sq(kstring_t *s, char *seq, int l, int rev, int comp)
 {
+	extern unsigned char seq_comp_table[256];
 	if (rev) {
 		int i;
 		str_enlarge(s, l);
 		for (i = 0; i < l; ++i) {
 			int c = seq[l - 1 - i];
-			s->s[s->l + i] = c < 128 && comp? comp_tab[c] : c;
+			s->s[s->l + i] = c < 128 && comp? seq_comp_table[c] : c;
 		}
 		s->l += l;
 	} else str_copy(s, seq, seq + l);
 }
 
-void mm_write_sam(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const mm_reg1_t *r, int n_regs, const mm_reg1_t *regs)
+static inline const mm_reg1_t *get_sam_pri(int n_regs, const mm_reg1_t *regs)
 {
-	int flag = 0;
+	int i;
+	for (i = 0; i < n_regs; ++i)
+		if (regs[i].sam_pri)
+			return &regs[i];
+	assert(n_regs == 0);
+	return NULL;
+}
+
+static void write_sam_cigar(kstring_t *s, int sam_flag, int in_tag, int qlen, const mm_reg1_t *r, int opt_flag)
+{
+	if (r->p == 0) {
+		mm_sprintf_lite(s, "*");
+	} else {
+		uint32_t k, clip_len[2];
+		clip_len[0] = r->rev? qlen - r->qe : r->qs;
+		clip_len[1] = r->rev? r->qs : qlen - r->qe;
+		if (in_tag) {
+			int clip_char = (sam_flag&0x800) && !(opt_flag&MM_F_SOFTCLIP)? 5 : 4;
+			mm_sprintf_lite(s, "\tCG:B:I");
+			if (clip_len[0]) mm_sprintf_lite(s, ",%u", clip_len[0]<<4|clip_char);
+			for (k = 0; k < r->p->n_cigar; ++k)
+				mm_sprintf_lite(s, ",%u", r->p->cigar[k]);
+			if (clip_len[1]) mm_sprintf_lite(s, ",%u", clip_len[1]<<4|clip_char);
+		} else {
+			int clip_char = (sam_flag&0x800) && !(opt_flag&MM_F_SOFTCLIP)? 'H' : 'S';
+			if (clip_len[0]) mm_sprintf_lite(s, "%d%c", clip_len[0], clip_char);
+			for (k = 0; k < r->p->n_cigar; ++k)
+				mm_sprintf_lite(s, "%d%c", r->p->cigar[k]>>4, "MIDNSHP=XB"[r->p->cigar[k]&0xf]);
+			if (clip_len[1]) mm_sprintf_lite(s, "%d%c", clip_len[1], clip_char);
+		}
+	}
+}
+
+void mm_write_sam2(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, int seg_idx, int reg_idx, int n_seg, const int *n_regss, const mm_reg1_t *const* regss, void *km, int opt_flag)
+{
+	const int max_bam_cigar_op = 65535;
+	int flag, n_regs = n_regss[seg_idx], cigar_in_tag = 0;
+	int this_rid = -1, this_pos = -1, this_rev = 0;
+	const mm_reg1_t *regs = regss[seg_idx], *r_prev = NULL, *r_next;
+	const mm_reg1_t *r = n_regs > 0 && reg_idx < n_regs && reg_idx >= 0? &regs[reg_idx] : NULL;
+
+	// find the primary of the previous and the next segments, if they are mapped
+	if (n_seg > 1) {
+		int i, next_sid = (seg_idx + 1) % n_seg;
+		r_next = get_sam_pri(n_regss[next_sid], regss[next_sid]);
+		if (n_seg > 2) {
+			for (i = 1; i <= n_seg - 1; ++i) {
+				int prev_sid = (seg_idx + n_seg - i) % n_seg;
+				if (n_regss[prev_sid] > 0) {
+					r_prev = get_sam_pri(n_regss[prev_sid], regss[prev_sid]);
+					break;
+				}
+			}
+		} else r_prev = r_next;
+	} else r_prev = r_next = NULL;
+
+	// write QNAME
 	s->l = 0;
+	mm_sprintf_lite(s, "%s", t->name);
+	if (n_seg > 1) s->l = mm_qname_len(t->name); // trim the suffix like /1 or /2
+
+	// write flag
+	flag = n_seg > 1? 0x1 : 0x0;
+	if (r == 0) {
+		flag |= 0x4;
+	} else {
+		if (r->rev) flag |= 0x10;
+		if (r->parent != r->id) flag |= 0x100;
+		else if (!r->sam_pri) flag |= 0x800;
+	}
+	if (n_seg > 1) {
+		if (r && r->proper_frag) flag |= 0x2; // TODO: this doesn't work when there are more than 2 segments
+		if (seg_idx == 0) flag |= 0x40;
+		else if (seg_idx == n_seg - 1) flag |= 0x80;
+		if (r_next == NULL) flag |= 0x8;
+		else if (r_next->rev) flag |= 0x20;
+	}
+	mm_sprintf_lite(s, "\t%d", flag);
+
+	// write coordinate, MAPQ and CIGAR
+	if (r == 0) {
+		if (r_prev) {
+			this_rid = r_prev->rid, this_pos = r_prev->rs;
+			mm_sprintf_lite(s, "\t%s\t%d\t0\t*", mi->seq[this_rid].name, this_pos+1);
+		} else mm_sprintf_lite(s, "\t*\t0\t0\t*");
+	} else {
+		this_rid = r->rid, this_pos = r->rs, this_rev = r->rev;
+		mm_sprintf_lite(s, "\t%s\t%d\t%d\t", mi->seq[r->rid].name, r->rs+1, r->mapq);
+		if ((opt_flag & MM_F_LONG_CIGAR) && r->p && r->p->n_cigar > max_bam_cigar_op - 2) {
+			int n_cigar = r->p->n_cigar;
+			if (r->qs != 0) ++n_cigar;
+			if (r->qe != t->l_seq) ++n_cigar;
+			if (n_cigar > max_bam_cigar_op)
+				cigar_in_tag = 1;
+		}
+		if (cigar_in_tag) {
+			int slen;
+			if ((flag & 0x900) == 0 || (opt_flag & MM_F_SOFTCLIP)) slen = t->l_seq;
+			else if (flag & 0x100) slen = 0;
+			else slen = r->qe - r->qs;
+			mm_sprintf_lite(s, "%dS%dN", slen, r->re - r->rs);
+		} else write_sam_cigar(s, flag, 0, t->l_seq, r, opt_flag);
+	}
+
+	// write mate positions
+	if (n_seg > 1) {
+		int tlen = 0;
+		if (this_rid >= 0 && r_next) {
+			if (this_rid == r_next->rid) {
+				int this_pos5 = r && r->rev? r->re - 1 : this_pos;
+				int next_pos5 = r_next->rev? r_next->re - 1 : r_next->rs;
+				tlen = next_pos5 - this_pos5;
+				mm_sprintf_lite(s, "\t=\t");
+			} else mm_sprintf_lite(s, "\t%s\t", mi->seq[r_next->rid].name);
+			mm_sprintf_lite(s, "%d\t", r_next->rs + 1);
+		} else if (r_next) { // && this_rid < 0
+			mm_sprintf_lite(s, "\t%s\t%d\t", mi->seq[r_next->rid].name, r_next->rs + 1);
+		} else if (this_rid >= 0) { // && r_next == NULL
+			int this_pos5 = this_rev? r->re - 1 : this_pos; // this_rev is only true when r != NULL
+			tlen = this_pos - this_pos5; // next_pos5 will be this_pos
+			mm_sprintf_lite(s, "\t=\t%d\t", this_pos + 1); // next segment will take r's coordinate
+		} else mm_sprintf_lite(s, "\t*\t0\t"); // neither has coordinates
+		if (tlen > 0) ++tlen;
+		else if (tlen < 0) --tlen;
+		mm_sprintf_lite(s, "%d\t", tlen);
+	} else mm_sprintf_lite(s, "\t*\t0\t0\t");
+
+	// write SEQ and QUAL
 	if (r == 0) {
-		mm_sprintf_lite(s, "%s\t4\t*\t0\t0\t*\t*\t0\t0\t", t->name);
 		sam_write_sq(s, t->seq, t->l_seq, 0, 0);
 		mm_sprintf_lite(s, "\t");
 		if (t->qual) sam_write_sq(s, t->qual, t->l_seq, 0, 0);
 		else mm_sprintf_lite(s, "*");
 	} else {
-		if (r->rev) flag |= 0x10;
-		if (r->parent != r->id) flag |= 0x100;
-		else if (!r->sam_pri) flag |= 0x800;
-		mm_sprintf_lite(s, "%s\t%d\t%s\t%d\t%d\t", t->name, flag, mi->seq[r->rid].name, r->rs+1, r->mapq);
-		if (r->p) { // actually this should always be true for SAM output
-			uint32_t k, clip_len = r->rev? t->l_seq - r->qe : r->qs;
-			int clip_char = (flag&0x800)? 'H' : 'S';
-			if (clip_len) mm_sprintf_lite(s, "%d%c", clip_len, clip_char);
-			for (k = 0; k < r->p->n_cigar; ++k)
-				mm_sprintf_lite(s, "%d%c", r->p->cigar[k]>>4, "MIDN"[r->p->cigar[k]&0xf]);
-			clip_len = r->rev? r->qs : t->l_seq - r->qe;
-			if (clip_len) mm_sprintf_lite(s, "%d%c", clip_len, clip_char);
-		} else mm_sprintf_lite(s, "*");
-		mm_sprintf_lite(s, "\t*\t0\t0\t");
-		if ((flag & 0x900) == 0) {
+		if ((flag & 0x900) == 0 || (opt_flag & MM_F_SOFTCLIP)) {
 			sam_write_sq(s, t->seq, t->l_seq, r->rev, r->rev);
 			mm_sprintf_lite(s, "\t");
 			if (t->qual) sam_write_sq(s, t->qual, t->l_seq, r->rev, 0);
@@ -282,8 +470,13 @@ void mm_write_sam(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const m
 			if (t->qual) sam_write_sq(s, t->qual + r->qs, r->qe - r->qs, r->rev, 0);
 			else mm_sprintf_lite(s, "*");
 		}
+	}
+
+	// write tags
+	if (mm_rg_id[0]) mm_sprintf_lite(s, "\tRG:Z:%s", mm_rg_id);
+	if (n_seg > 2) mm_sprintf_lite(s, "\tFI:i:%d", seg_idx);
+	if (r) {
 		write_tags(s, r);
-		if (mm_rg_id[0]) mm_sprintf_lite(s, "\tRG:Z:%s", mm_rg_id);
 		if (r->parent == r->id && r->p && n_regs > 1 && regs && r >= regs && r - regs < n_regs) { // supplementary aln may exist
 			int i, n_sa = 0; // n_sa: number of SA fields
 			for (i = 0; i < n_regs; ++i)
@@ -305,10 +498,26 @@ void mm_write_sam(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const m
 					if (l_I) mm_sprintf_lite(s, "%dI", l_I);
 					if (l_D) mm_sprintf_lite(s, "%dD", l_D);
 					if (clip3) mm_sprintf_lite(s, "%dS", clip3);
-					mm_sprintf_lite(s, ",%d,%d;", q->mapq, q->p->n_diff);
+					mm_sprintf_lite(s, ",%d,%d;", q->mapq, q->blen - q->mlen + q->p->n_ambi);
 				}
 			}
 		}
+		if (r->p && (opt_flag & (MM_F_OUT_CS|MM_F_OUT_MD)))
+			write_cs_or_MD(km, s, mi, t, r, !(opt_flag&MM_F_OUT_CS_LONG), opt_flag&MM_F_OUT_MD, 1);
+		if (cigar_in_tag)
+			write_sam_cigar(s, flag, 1, t->l_seq, r, opt_flag);
 	}
+
+	if ((opt_flag & MM_F_COPY_COMMENT) && t->comment)
+		mm_sprintf_lite(s, "\t%s", t->comment);
+
 	s->s[s->l] = 0; // we always have room for an extra byte (see str_enlarge)
 }
+
+void mm_write_sam(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const mm_reg1_t *r, int n_regs, const mm_reg1_t *regs)
+{
+	int i;
+	for (i = 0; i < n_regs; ++i)
+		if (r == &regs[i]) break;
+	mm_write_sam2(s, mi, t, 0, i, 1, &n_regs, &regs, NULL, 0);
+}

hit.c

@@ -1,20 +1,22 @@
 #include <string.h>
+#include <stdlib.h>
 #include <math.h>
 #include "mmpriv.h"
 #include "kalloc.h"
+#include "khash.h"
 
 static inline void mm_cal_fuzzy_len(mm_reg1_t *r, const mm128_t *a)
 {
 	int i;
-	r->fuzzy_mlen = r->fuzzy_blen = 0;
+	r->mlen = r->blen = 0;
 	if (r->cnt <= 0) return;
-	r->fuzzy_mlen = r->fuzzy_blen = a[r->as].y>>32&0xff;
+	r->mlen = r->blen = a[r->as].y>>32&0xff;
 	for (i = r->as + 1; i < r->as + r->cnt; ++i) {
 		int span = a[i].y>>32&0xff;
 		int tl = (int32_t)a[i].x - (int32_t)a[i-1].x;
 		int ql = (int32_t)a[i].y - (int32_t)a[i-1].y;
-		r->fuzzy_blen += tl > ql? tl : ql;
-		r->fuzzy_mlen += tl > span && ql > span? span : tl < ql? tl : ql;
+		r->blen += tl > ql? tl : ql;
+		r->mlen += tl > span && ql > span? span : tl < ql? tl : ql;
 	}
 }
 
@@ -35,7 +37,19 @@ static inline void mm_reg_set_coor(mm_reg1_t *r, int32_t qlen, const mm128_t *a)
 	mm_cal_fuzzy_len(r, a);
 }
 
-mm_reg1_t *mm_gen_regs(void *km, int qlen, int n_u, uint64_t *u, mm128_t *a) // convert chains to hits
+static inline uint64_t hash64(uint64_t key)
+{
+	key = (~key + (key << 21));
+	key = key ^ key >> 24;
+	key = ((key + (key << 3)) + (key << 8));
+	key = key ^ key >> 14;
+	key = ((key + (key << 2)) + (key << 4));
+	key = key ^ key >> 28;
+	key = (key + (key << 31));
+	return key;
+}
+
+mm_reg1_t *mm_gen_regs(void *km, uint32_t hash, int qlen, int n_u, uint64_t *u, mm128_t *a) // convert chains to hits
 {
 	mm128_t *z, tmp;
 	mm_reg1_t *r;
@@ -46,7 +60,9 @@ mm_reg1_t *mm_gen_regs(void *km, int qlen, int n_u, uint64_t *u, mm128_t *a) //
 	// sort by score
 	z = (mm128_t*)kmalloc(km, n_u * 16);
 	for (i = k = 0; i < n_u; ++i) {
-		z[i].x = u[i] >> 32;
+		uint32_t h;
+		h = (uint32_t)hash64((hash64(a[k].x) + hash64(a[k].y)) ^ hash);
+		z[i].x = u[i] ^ h; // u[i] -- higher 32 bits: chain score; lower 32 bits: number of seeds in the chain
 		z[i].y = (uint64_t)k << 32 | (int32_t)u[i];
 		k += (int32_t)u[i];
 	}
@@ -60,9 +76,11 @@ mm_reg1_t *mm_gen_regs(void *km, int qlen, int n_u, uint64_t *u, mm128_t *a) //
 		mm_reg1_t *ri = &r[i];
 		ri->id = i;
 		ri->parent = MM_PARENT_UNSET;
-		ri->score = z[i].x;
+		ri->score = ri->score0 = z[i].x >> 32;
+		ri->hash = (uint32_t)z[i].x;
 		ri->cnt = (int32_t)z[i].y;
 		ri->as = z[i].y >> 32;
+		ri->div = -1.0f;
 		mm_reg_set_coor(ri, qlen, a);
 	}
 	kfree(km, z);
@@ -76,6 +94,7 @@ void mm_split_reg(mm_reg1_t *r, mm_reg1_t *r2, int n, int qlen, mm128_t *a)
 	r2->id = -1;
 	r2->sam_pri = 0;
 	r2->p = 0;
+	r2->split_inv = 0;
 	r2->cnt = r->cnt - n;
 	r2->score = (int32_t)(r->score * ((float)r2->cnt / r->cnt) + .499);
 	r2->as = r->as + n;
@@ -87,55 +106,94 @@ void mm_split_reg(mm_reg1_t *r, mm_reg1_t *r2, int n, int qlen, mm128_t *a)
 	r->split |= 1, r2->split |= 2;
 }
 
-void mm_set_parent(void *km, float mask_level, int n, mm_reg1_t *r) // and compute mm_reg1_t::subsc
+void mm_set_parent(void *km, float mask_level, int n, mm_reg1_t *r, int sub_diff, int hard_mask_level) // and compute mm_reg1_t::subsc
 {
 	int i, j, k, *w;
+	uint64_t *cov;
 	if (n <= 0) return;
 	for (i = 0; i < n; ++i) r[i].id = i;
+	cov = (uint64_t*)kmalloc(km, n * sizeof(uint64_t));
 	w = (int*)kmalloc(km, n * sizeof(int));
 	w[0] = 0, r[0].parent = 0;
 	for (i = 1, k = 1; i < n; ++i) {
 		mm_reg1_t *ri = &r[i];
-		int si = ri->qs, ei = ri->qe;
-		for (j = 0; j < k; ++j) {
+		int si = ri->qs, ei = ri->qe, n_cov = 0, uncov_len = 0;
+		if (hard_mask_level) goto skip_uncov;
+		for (j = 0; j < k; ++j) { // traverse existing primary hits to find overlapping hits
 			mm_reg1_t *rp = &r[w[j]];
 			int sj = rp->qs, ej = rp->qe;
-			int min = ej - sj < ei - si? ej - sj : ei - si;
-			int ol = si < sj? (ei < sj? 0 : ei < ej? ei - sj : ej - sj) : (ej < si? 0 : ej < ei? ej - si : ei - si);
-			if (ol > mask_level * min) {
+			if (ej <= si || sj >= ei) continue;
+			if (sj < si) sj = si;
+			if (ej > ei) ej = ei;
+			cov[n_cov++] = (uint64_t)sj<<32 | ej;
+		}
+		if (n_cov == 0) {
+			goto set_parent_test; // no overlapping primary hits; then i is a new primary hit
+		} else if (n_cov > 0) { // there are overlapping primary hits; find the length not covered by existing primary hits
+			int j, x = si;
+			radix_sort_64(cov, cov + n_cov);
+			for (j = 0; j < n_cov; ++j) {
+				if ((int)(cov[j]>>32) > x) uncov_len += (cov[j]>>32) - x;
+				x = (int32_t)cov[j] > x? (int32_t)cov[j] : x;
+			}
+			if (ei > x) uncov_len += ei - x;
+		}
+skip_uncov:
+		for (j = 0; j < k; ++j) { // traverse existing primary hits again
+			mm_reg1_t *rp = &r[w[j]];
+			int sj = rp->qs, ej = rp->qe, min, max, ol;
+			if (ej <= si || sj >= ei) continue; // no overlap
+			min = ej - sj < ei - si? ej - sj : ei - si;
+			max = ej - sj > ei - si? ej - sj : ei - si;
+			ol = si < sj? (ei < sj? 0 : ei < ej? ei - sj : ej - sj) : (ej < si? 0 : ej < ei? ej - si : ei - si); // overlap length; TODO: this can be simplified
+			if ((float)ol / min - (float)uncov_len / max > mask_level) {
+				int cnt_sub = 0;
 				ri->parent = rp->parent;
 				rp->subsc = rp->subsc > ri->score? rp->subsc : ri->score;
-				if (rp->p && ri->p)
+				if (ri->cnt >= rp->cnt) cnt_sub = 1;
+				if (rp->p && ri->p && (rp->rid != ri->rid || rp->rs != ri->rs || rp->re != ri->re || ol != min)) { // the last condition excludes identical hits after DP
 					rp->p->dp_max2 = rp->p->dp_max2 > ri->p->dp_max? rp->p->dp_max2 : ri->p->dp_max;
+					if (rp->p->dp_max - ri->p->dp_max <= sub_diff) cnt_sub = 1;
+				}
+				if (cnt_sub) ++rp->n_sub;
 				break;
 			}
 		}
-		if (j == k) w[k++] = i, ri->parent = i;
+set_parent_test:
+		if (j == k) w[k++] = i, ri->parent = i, ri->n_sub = 0;
 	}
+	kfree(km, cov);
 	kfree(km, w);
 }
 
-void mm_hit_sort_by_dp(void *km, int *n_regs, mm_reg1_t *r)
+void mm_hit_sort(void *km, int *n_regs, mm_reg1_t *r)
 {
-	int32_t i, n_aux, n = *n_regs;
-	uint64_t *aux;
+	int32_t i, n_aux, n = *n_regs, has_cigar = 0, no_cigar = 0;
+	mm128_t *aux;
 	mm_reg1_t *t;
 
 	if (n <= 1) return;
-	aux = (uint64_t*)kmalloc(km, n * 8);
+	aux = (mm128_t*)kmalloc(km, n * 16);
 	t = (mm_reg1_t*)kmalloc(km, n * sizeof(mm_reg1_t));
 	for (i = n_aux = 0; i < n; ++i) {
 		if (r[i].inv || r[i].cnt > 0) { // squeeze out elements with cnt==0 (soft deleted)
-			assert(r[i].p);
-			aux[n_aux++] = (uint64_t)r[i].p->dp_max << 32 | i;
+			if (r[i].p) {
+				aux[n_aux].x = (uint64_t)r[i].p->dp_max << 32 | r[i].hash;
+				has_cigar = 1;
+			} else {
+				aux[n_aux].x = (uint64_t)r[i].score << 32 | r[i].hash;
+				no_cigar = 1;
+			}
+			aux[n_aux++].y = i;
 		} else if (r[i].p) {
 			free(r[i].p);
 			r[i].p = 0;
 		}
 	}
-	radix_sort_64(aux, aux + n_aux);
+	assert(has_cigar + no_cigar == 1);
+	radix_sort_128x(aux, aux + n_aux);
 	for (i = n_aux - 1; i >= 0; --i)
-		t[n_aux - 1 - i] = r[(int32_t)aux[i]];
+		t[n_aux - 1 - i] = r[aux[i].y];
 	memcpy(r, t, sizeof(mm_reg1_t) * n_aux);
 	*n_regs = n_aux;
 	kfree(km, aux);
@@ -177,30 +235,36 @@ void mm_sync_regs(void *km, int n_regs, mm_reg1_t *regs) // keep mm_reg1_t::{id,
 	mm_set_sam_pri(n_regs, regs);
 }
 
-void mm_select_sub(void *km, float mask_level, float pri_ratio, int min_diff, int best_n, int *n_, mm_reg1_t *r)
+void mm_select_sub(void *km, float pri_ratio, int min_diff, int best_n, int *n_, mm_reg1_t *r)
 {
 	if (pri_ratio > 0.0f && *n_ > 0) {
 		int i, k, n = *n_, n_2nd = 0;
-		for (i = k = 0; i < n; ++i)
-			if (r[i].parent == i) r[k++] = r[i];
-			else if ((r[i].score >= r[r[i].parent].score * pri_ratio || r[i].score + min_diff >= r[r[i].parent].score) && n_2nd++ < best_n)
+		for (i = k = 0; i < n; ++i) {
+			int p = r[i].parent;
+			if (p == i || r[i].inv) { // primary or inversion
 				r[k++] = r[i];
-			else if (r[i].p) free(r[i].p);
+			} else if ((r[i].score >= r[p].score * pri_ratio || r[i].score + min_diff >= r[p].score) && n_2nd < best_n) {
+				if (!(r[i].qs == r[p].qs && r[i].qe == r[p].qe && r[i].rid == r[p].rid && r[i].rs == r[p].rs && r[i].re == r[p].re)) // not identical hits
+					r[k++] = r[i], ++n_2nd;
+				else if (r[i].p) free(r[i].p);
+			} else if (r[i].p) free(r[i].p);
+		}
 		if (k != n) mm_sync_regs(km, k, r); // removing hits requires sync()
 		*n_ = k;
 	}
 }
 
-void mm_filter_regs(void *km, const mm_mapopt_t *opt, int *n_regs, mm_reg1_t *regs)
+void mm_filter_regs(const mm_mapopt_t *opt, int qlen, int *n_regs, mm_reg1_t *regs)
 { // NB: after this call, mm_reg1_t::parent can be -1 if its parent filtered out
 	int i, k;
 	for (i = k = 0; i < *n_regs; ++i) {
 		mm_reg1_t *r = &regs[i];
 		int flt = 0;
-		if (!r->inv && r->cnt < opt->min_cnt) flt = 1;
-		if (r->p) {
-			if (r->p->blen - r->p->n_ambi - r->p->n_diff < opt->min_chain_score) flt = 1;
+		if (!r->inv && !r->seg_split && r->cnt < opt->min_cnt) flt = 1;
+		if (r->p) { // these filters are only applied when base-alignment is available
+			if (r->mlen < opt->min_chain_score) flt = 1;
 			else if (r->p->dp_max < opt->min_dp_max) flt = 1;
+			else if (r->qs > qlen * opt->max_clip_ratio && qlen - r->qe > qlen * opt->max_clip_ratio) flt = 1;
 			if (flt) free(r->p);
 		}
 		if (!flt) {
@@ -248,7 +312,7 @@ void mm_join_long(void *km, const mm_mapopt_t *opt, int qlen, int *n_regs_, mm_r
 	for (i = n_aux - 1; i >= 1; --i) {
 		mm_reg1_t *r0 = &regs[(int32_t)aux[i-1]], *r1 = &regs[(int32_t)aux[i]];
 		mm128_t *a0e, *a1s;
-		int max_gap, min_gap, sc_thres;
+		int max_gap, min_gap, sc_thres, min_flank_len;
 
 		// test
 		if (r0->as + r0->cnt != r1->as) continue; // not adjacent in a[]
@@ -257,13 +321,14 @@ void mm_join_long(void *km, const mm_mapopt_t *opt, int qlen, int *n_regs_, mm_r
 		a1s = &a[r1->as];
 		if (a1s->x <= a0e->x || (int32_t)a1s->y <= (int32_t)a0e->y) continue; // keep colinearity
 		max_gap = min_gap = (int32_t)a1s->y - (int32_t)a0e->y;
-		max_gap = max_gap > a1s->x - a0e->x? max_gap : a1s->x - a0e->x;
-		min_gap = min_gap < a1s->x - a0e->x? min_gap : a1s->x - a0e->x;
+		max_gap = a0e->x + max_gap > a1s->x? max_gap : a1s->x - a0e->x;
+		min_gap = a0e->x + min_gap < a1s->x? min_gap : a1s->x - a0e->x;
 		if (max_gap > opt->max_join_long || min_gap > opt->max_join_short) continue;
 		sc_thres = (int)((float)opt->min_join_flank_sc / opt->max_join_long * max_gap + .499);
 		if (r0->score < sc_thres || r1->score < sc_thres) continue; // require good flanking chains
-		if (r0->re - r0->rs < max_gap>>1 || r0->qe - r0->qs < max_gap>>1) continue; // require enough flanking length
-		if (r1->re - r1->rs < max_gap>>1 || r1->qe - r1->qs < max_gap>>1) continue;
+		min_flank_len = (int)(max_gap * opt->min_join_flank_ratio);
+		if (r0->re - r0->rs < min_flank_len || r0->qe - r0->qs < min_flank_len) continue; // require enough flanking length
+		if (r1->re - r1->rs < min_flank_len || r1->qe - r1->qs < min_flank_len) continue;
 
 		// all conditions satisfied; join
 		a[r1->as].y |= MM_SEED_LONG_JOIN;
@@ -283,29 +348,143 @@ void mm_join_long(void *km, const mm_mapopt_t *opt, int qlen, int *n_regs_, mm_r
 					r->parent = regs[r->parent].parent;
 			}
 		}
-		mm_filter_regs(km, opt, n_regs_, regs);
+		mm_filter_regs(opt, qlen, n_regs_, regs);
 		mm_sync_regs(km, *n_regs_, regs);
 	}
 }
 
-void mm_set_mapq(int n_regs, mm_reg1_t *regs, int min_chain_sc)
+mm_seg_t *mm_seg_gen(void *km, uint32_t hash, int n_segs, const int *qlens, int n_regs0, const mm_reg1_t *regs0, int *n_regs, mm_reg1_t **regs, const mm128_t *a)
+{
+	int s, i, j, acc_qlen[MM_MAX_SEG+1], qlen_sum = 0;
+	mm_seg_t *seg;
+
+	assert(n_segs <= MM_MAX_SEG);
+	for (s = 1, acc_qlen[0] = 0; s < n_segs; ++s)
+		acc_qlen[s] = acc_qlen[s-1] + qlens[s-1];
+	qlen_sum = acc_qlen[n_segs - 1] + qlens[n_segs - 1];
+
+	seg = (mm_seg_t*)kcalloc(km, n_segs, sizeof(mm_seg_t));
+	for (s = 0; s < n_segs; ++s) {
+		seg[s].u = (uint64_t*)kmalloc(km, n_regs0 * 8);
+		for (i = 0; i < n_regs0; ++i)
+			seg[s].u[i] = (uint64_t)regs0[i].score << 32;
+	}
+	for (i = 0; i < n_regs0; ++i) {
+		const mm_reg1_t *r = &regs0[i];
+		for (j = 0; j < r->cnt; ++j) {
+			int sid = (a[r->as + j].y&MM_SEED_SEG_MASK)>>MM_SEED_SEG_SHIFT;
+			++seg[sid].u[i];
+			++seg[sid].n_a;
+		}
+	}
+	for (s = 0; s < n_segs; ++s) {
+		mm_seg_t *sr = &seg[s];
+		for (i = 0, sr->n_u = 0; i < n_regs0; ++i) // squeeze out zero-length per-segment chains
+			if ((int32_t)sr->u[i] != 0)
+				sr->u[sr->n_u++] = sr->u[i];
+		sr->a = (mm128_t*)kmalloc(km, sr->n_a * sizeof(mm128_t));
+		sr->n_a = 0;
+	}
+
+	for (i = 0; i < n_regs0; ++i) {
+		const mm_reg1_t *r = &regs0[i];
+		for (j = 0; j < r->cnt; ++j) {
+			int sid = (a[r->as + j].y&MM_SEED_SEG_MASK)>>MM_SEED_SEG_SHIFT;
+			mm128_t a1 = a[r->as + j];
+			// on reverse strand, the segment position is:
+			//   x_for_cat = qlen_sum - 1 - (int32_t)a1.y - 1 + q_span
+			//   (int32_t)new_a1.y = qlens[sid] - (x_for_cat - acc_qlen[sid] + 1 - q_span) - 1 = (int32_t)a1.y - (qlen_sum - (qlens[sid] + acc_qlen[sid]))
+			a1.y -= a1.x>>63? qlen_sum - (qlens[sid] + acc_qlen[sid]) : acc_qlen[sid];
+			seg[sid].a[seg[sid].n_a++] = a1;
+		}
+	}
+	for (s = 0; s < n_segs; ++s) {
+		regs[s] = mm_gen_regs(km, hash, qlens[s], seg[s].n_u, seg[s].u, seg[s].a);
+		n_regs[s] = seg[s].n_u;
+		for (i = 0; i < n_regs[s]; ++i) {
+			regs[s][i].seg_split = 1;
+			regs[s][i].seg_id = s;
+		}
+	}
+	return seg;
+}
+
+void mm_seg_free(void *km, int n_segs, mm_seg_t *segs)
 {
-	static const float q_coef = 30.0f;
 	int i;
+	for (i = 0; i < n_segs; ++i) kfree(km, segs[i].u);
+	for (i = 0; i < n_segs; ++i) kfree(km, segs[i].a);
+	kfree(km, segs);
+}
+
+static void mm_set_inv_mapq(void *km, int n_regs, mm_reg1_t *regs)
+{
+	int i, n_aux;
+	mm128_t *aux;
+	if (n_regs < 3) return;
+	for (i = 0; i < n_regs; ++i)
+		if (regs[i].inv) break;
+	if (i == n_regs) return; // no inversion hits
+
+	aux = (mm128_t*)kmalloc(km, n_regs * 16);
+	for (i = n_aux = 0; i < n_regs; ++i)
+		if (regs[i].parent == i || regs[i].parent < 0)
+			aux[n_aux].y = i, aux[n_aux++].x = (uint64_t)regs[i].rid << 32 | regs[i].rs;
+	radix_sort_128x(aux, aux + n_aux);
+
+	for (i = 1; i < n_aux - 1; ++i) {
+		mm_reg1_t *inv = &regs[aux[i].y];
+		if (inv->inv) {
+			mm_reg1_t *l = &regs[aux[i-1].y];
+			mm_reg1_t *r = &regs[aux[i+1].y];
+			inv->mapq = l->mapq < r->mapq? l->mapq : r->mapq;
+		}
+	}
+	kfree(km, aux);
+}
+
+void mm_set_mapq(void *km, int n_regs, mm_reg1_t *regs, int min_chain_sc, int match_sc, int rep_len, int is_sr)
+{
+	static const float q_coef = 40.0f;
+	int64_t sum_sc = 0;
+	float uniq_ratio;
+	int i;
+	for (i = 0; i < n_regs; ++i)
+		if (regs[i].parent == regs[i].id)
+			sum_sc += regs[i].score;
+	uniq_ratio = (float)sum_sc / (sum_sc + rep_len);
 	for (i = 0; i < n_regs; ++i) {
 		mm_reg1_t *r = &regs[i];
 		if (r->inv) {
 			r->mapq = 0;
 		} else if (r->parent == r->id) {
 			int mapq, subsc;
-			float pen_cm = r->cnt >= 10? 1.0f : 0.1f * r->cnt;
+			float pen_s1 = (r->score > 100? 1.0f : 0.01f * r->score) * uniq_ratio;
+			float pen_cm = r->cnt > 10? 1.0f : 0.1f * r->cnt;
+			pen_cm = pen_s1 < pen_cm? pen_s1 : pen_cm;
 			subsc = r->subsc > min_chain_sc? r->subsc : min_chain_sc;
 			if (r->p && r->p->dp_max2 > 0 && r->p->dp_max > 0) {
-				float identity = (float)(r->p->blen - r->p->n_diff - r->p->n_ambi) / (r->p->blen - r->p->n_ambi);
-				mapq = (int)(identity * pen_cm * q_coef * (1. - (float)r->p->dp_max2 * subsc / r->p->dp_max / r->score) * logf(r->score));
-			} else mapq = (int)(pen_cm * q_coef * (1. - (float)subsc / r->score) * logf(r->score));
+				float identity = (float)r->mlen / r->blen;
+				float x = (float)r->p->dp_max2 * subsc / r->p->dp_max / r->score0;
+				mapq = (int)(identity * pen_cm * q_coef * (1.0f - x * x) * logf((float)r->p->dp_max / match_sc));
+				if (!is_sr) {
+					int mapq_alt = (int)(6.02f * identity * identity * (r->p->dp_max - r->p->dp_max2) / match_sc + .499f); // BWA-MEM like mapQ, mostly for short reads
+					mapq = mapq < mapq_alt? mapq : mapq_alt; // in case the long-read heuristic fails
+				}
+			} else {
+				float x = (float)subsc / r->score0;
+				if (r->p) {
+					float identity = (float)r->mlen / r->blen;
+					mapq = (int)(identity * pen_cm * q_coef * (1.0f - x) * logf((float)r->p->dp_max / match_sc));
+				} else {
+					mapq = (int)(pen_cm * q_coef * (1.0f - x) * logf(r->score));
+				}
+			}
+			mapq -= (int)(4.343f * logf(r->n_sub + 1) + .499f);
 			mapq = mapq > 0? mapq : 0;
 			r->mapq = mapq < 60? mapq : 60;
+			if (r->p && r->p->dp_max > r->p->dp_max2 && r->mapq == 0) r->mapq = 1;
 		} else r->mapq = 0;
 	}
+	mm_set_inv_mapq(km, n_regs, regs);
 }

index.c

@@ -1,8 +1,13 @@
 #include <stdlib.h>
 #include <assert.h>
+#if defined(WIN32) || defined(_WIN32)
+#include <io.h> // for open(2)
+#else
 #include <unistd.h>
+#endif
 #include <fcntl.h>
 #include <stdio.h>
+#define __STDC_LIMIT_MACROS
 #include "kthread.h"
 #include "bseq.h"
 #include "minimap.h"
@@ -15,15 +20,24 @@
 KHASH_INIT(idx, uint64_t, uint64_t, 1, idx_hash, idx_eq)
 typedef khash_t(idx) idxhash_t;
 
+KHASH_MAP_INIT_STR(str, uint32_t)
+
 #define kroundup64(x) (--(x), (x)|=(x)>>1, (x)|=(x)>>2, (x)|=(x)>>4, (x)|=(x)>>8, (x)|=(x)>>16, (x)|=(x)>>32, ++(x))
 
-mm_idx_t *mm_idx_init(int w, int k, int b, int is_hpc)
+typedef struct mm_idx_bucket_s {
+	mm128_v a;   // (minimizer, position) array
+	int32_t n;   // size of the _p_ array
+	uint64_t *p; // position array for minimizers appearing >1 times
+	void *h;     // hash table indexing _p_ and minimizers appearing once
+} mm_idx_bucket_t;
+
+mm_idx_t *mm_idx_init(int w, int k, int b, int flag)
 {
 	mm_idx_t *mi;
 	if (k*2 < b) b = k * 2;
 	if (w < 1) w = 1;
 	mi = (mm_idx_t*)calloc(1, sizeof(mm_idx_t));
-	mi->w = w, mi->k = k, mi->b = b, mi->is_hpc = is_hpc;
+	mi->w = w, mi->k = k, mi->b = b, mi->flag = flag;
 	mi->B = (mm_idx_bucket_t*)calloc(1<<b, sizeof(mm_idx_bucket_t));
 	if (!(mm_dbg_flag & 1)) mi->km = km_init();
 	return mi;
@@ -31,12 +45,15 @@ mm_idx_t *mm_idx_init(int w, int k, int b, int is_hpc)
 
 void mm_idx_destroy(mm_idx_t *mi)
 {
-	int i;
+	uint32_t i;
 	if (mi == 0) return;
-	for (i = 0; i < 1<<mi->b; ++i) {
-		free(mi->B[i].p);
-		free(mi->B[i].a.a);
-		kh_destroy(idx, (idxhash_t*)mi->B[i].h);
+	if (mi->h) kh_destroy(str, (khash_t(str)*)mi->h);
+	if (mi->B) {
+		for (i = 0; i < 1U<<mi->b; ++i) {
+			free(mi->B[i].p);
+			free(mi->B[i].a.a);
+			kh_destroy(idx, (idxhash_t*)mi->B[i].h);
+		}
 	}
 	if (!mi->km) {
 		for (i = 0; i < mi->n_seq; ++i)
@@ -67,14 +84,15 @@ const uint64_t *mm_idx_get(const mm_idx_t *mi, uint64_t minier, int *n)
 
 void mm_idx_stat(const mm_idx_t *mi)
 {
-	int i, n = 0, n1 = 0;
+	int n = 0, n1 = 0;
+	uint32_t i;
 	uint64_t sum = 0, len = 0;
-	fprintf(stderr, "[M::%s] kmer size: %d; skip: %d; is_HPC: %d; #seq: %d\n", __func__, mi->k, mi->w, mi->is_hpc, mi->n_seq);
+	fprintf(stderr, "[M::%s] kmer size: %d; skip: %d; is_hpc: %d; #seq: %d\n", __func__, mi->k, mi->w, mi->flag&MM_I_HPC, mi->n_seq);
 	for (i = 0; i < mi->n_seq; ++i)
 		len += mi->seq[i].len;
-	for (i = 0; i < 1<<mi->b; ++i)
+	for (i = 0; i < 1U<<mi->b; ++i)
 		if (mi->B[i].h) n += kh_size((idxhash_t*)mi->B[i].h);
-	for (i = 0; i < 1<<mi->b; ++i) {
+	for (i = 0; i < 1U<<mi->b; ++i) {
 		idxhash_t *h = (idxhash_t*)mi->B[i].h;
 		khint_t k;
 		if (h == 0) continue;
@@ -88,6 +106,34 @@ void mm_idx_stat(const mm_idx_t *mi)
 			__func__, realtime() - mm_realtime0, cputime() / (realtime() - mm_realtime0), n, 100.0*n1/n, (double)sum / n, (double)len / sum);
 }
 
+int mm_idx_index_name(mm_idx_t *mi)
+{
+	khash_t(str) *h;
+	uint32_t i;
+	int has_dup = 0, absent;
+	if (mi->h) return 0;
+	h = kh_init(str);
+	for (i = 0; i < mi->n_seq; ++i) {
+		khint_t k;
+		k = kh_put(str, h, mi->seq[i].name, &absent);
+		if (absent) kh_val(h, k) = i;
+		else has_dup = 1;
+	}
+	mi->h = h;
+	if (has_dup && mm_verbose >= 2)
+		fprintf(stderr, "[WARNING] some database sequences have identical sequence names\n");
+	return has_dup;
+}
+
+int mm_idx_name2id(const mm_idx_t *mi, const char *name)
+{
+	khash_t(str) *h = (khash_t(str)*)mi->h;
+	khint_t k;
+	if (h == 0) return -2;
+	k = kh_get(str, h, name);
+	return k == kh_end(h)? -1 : kh_val(h, k);
+}
+
 int mm_idx_getseq(const mm_idx_t *mi, uint32_t rid, uint32_t st, uint32_t en, uint8_t *seq)
 {
 	uint64_t i, st1, en1;
@@ -100,13 +146,13 @@ int mm_idx_getseq(const mm_idx_t *mi, uint32_t rid, uint32_t st, uint32_t en, ui
 	return en - st;
 }
 
-uint32_t mm_idx_cal_max_occ(const mm_idx_t *mi, float f)
+int32_t mm_idx_cal_max_occ(const mm_idx_t *mi, float f)
 {
 	int i;
 	size_t n = 0;
 	uint32_t thres;
 	khint_t *a, k;
-	if (f <= 0.) return UINT32_MAX;
+	if (f <= 0.) return INT32_MAX;
 	for (i = 0; i < 1<<mi->b; ++i)
 		if (mi->B[i].h) n += kh_size((idxhash_t*)mi->B[i].h);
 	a = (uint32_t*)malloc(n * 4);
@@ -129,7 +175,8 @@ uint32_t mm_idx_cal_max_occ(const mm_idx_t *mi, float f)
 
 static void worker_post(void *g, long i, int tid)
 {
-	int j, start_a, start_p, n, n_keys;
+	int n, n_keys;
+	size_t j, start_a, start_p;
 	idxhash_t *h;
 	mm_idx_t *mi = (mm_idx_t*)g;
 	mm_idx_bucket_t *b = &mi->B[i];
@@ -157,7 +204,7 @@ static void worker_post(void *g, long i, int tid)
 			int absent;
 			mm128_t *p = &b->a.a[j-1];
 			itr = kh_put(idx, h, p->x>>8>>mi->b<<1, &absent);
-			assert(absent && j - start_a == n);
+			assert(absent && j == start_a + n);
 			if (n == 1) {
 				kh_key(h, itr) |= 1;
 				kh_val(h, itr) = p->y;
@@ -173,10 +220,10 @@ static void worker_post(void *g, long i, int tid)
 		} else ++n;
 	}
 	b->h = h;
-	assert(b->n == start_p);
+	assert(b->n == (int32_t)start_p);
 
 	// deallocate and clear b->a
-	free(b->a.a);
+	kfree(0, b->a.a);
 	b->a.n = b->a.m = 0, b->a.a = 0;
 }
  
@@ -194,7 +241,7 @@ static void mm_idx_post(mm_idx_t *mi, int n_threads)
 #include "bseq.h"
 
 typedef struct {
-	int mini_batch_size, keep_name;
+	int mini_batch_size;
 	uint64_t batch_size, sum_len;
 	mm_bseq_file_t *fp;
 	mm_idx_t *mi;
@@ -226,7 +273,6 @@ static void *worker_pipeline(void *shared, int step, void *in)
 		s->seq = mm_bseq_read(p->fp, p->mini_batch_size, 0, &s->n_seq); // read a mini-batch
 		if (s->seq) {
 			uint32_t old_m, m;
-			uint64_t sum_len, old_max_len, max_len;
 			assert((uint64_t)p->mi->n_seq + s->n_seq <= UINT32_MAX); // to prevent integer overflow
 			// make room for p->mi->seq
 			old_m = p->mi->n_seq, m = p->mi->n_seq + s->n_seq;
@@ -234,30 +280,34 @@ static void *worker_pipeline(void *shared, int step, void *in)
 			if (old_m != m)
 				p->mi->seq = (mm_idx_seq_t*)krealloc(p->mi->km, p->mi->seq, m * sizeof(mm_idx_seq_t));
 			// make room for p->mi->S
-			for (i = 0, sum_len = 0; i < s->n_seq; ++i) sum_len += s->seq[i].l_seq;
-			old_max_len = (p->sum_len + 7) / 8;
-			max_len = (p->sum_len + sum_len + 7) / 8;
-			kroundup64(old_max_len); kroundup64(max_len);
-			if (old_max_len != max_len) {
-				p->mi->S = (uint32_t*)realloc(p->mi->S, max_len * 4);
-				memset(&p->mi->S[old_max_len], 0, 4 * (max_len - old_max_len));
+			if (!(p->mi->flag & MM_I_NO_SEQ)) {
+				uint64_t sum_len, old_max_len, max_len;
+				for (i = 0, sum_len = 0; i < s->n_seq; ++i) sum_len += s->seq[i].l_seq;
+				old_max_len = (p->sum_len + 7) / 8;
+				max_len = (p->sum_len + sum_len + 7) / 8;
+				kroundup64(old_max_len); kroundup64(max_len);
+				if (old_max_len != max_len) {
+					p->mi->S = (uint32_t*)realloc(p->mi->S, max_len * 4);
+					memset(&p->mi->S[old_max_len], 0, 4 * (max_len - old_max_len));
+				}
 			}
 			// populate p->mi->seq
 			for (i = 0; i < s->n_seq; ++i) {
 				mm_idx_seq_t *seq = &p->mi->seq[p->mi->n_seq];
 				uint32_t j;
-				if (p->keep_name) {
-					assert(strlen(s->seq[i].name) <= 254); // a long query name breaks BAM
+				if (!(p->mi->flag & MM_I_NO_NAME)) {
 					seq->name = (char*)kmalloc(p->mi->km, strlen(s->seq[i].name) + 1);
 					strcpy(seq->name, s->seq[i].name);
 				} else seq->name = 0;
 				seq->len = s->seq[i].l_seq;
 				seq->offset = p->sum_len;
 				// copy the sequence
-				for (j = 0; j < seq->len; ++j) { // TODO: this is not the fastest way, but let's first see if speed matters here
-					uint64_t o = p->sum_len + j;
-					int c = seq_nt4_table[(uint8_t)s->seq[i].seq[j]];
-					mm_seq4_set(p->mi->S, o, c);
+				if (!(p->mi->flag & MM_I_NO_SEQ)) {
+					for (j = 0; j < seq->len; ++j) { // TODO: this is not the fastest way, but let's first see if speed matters here
+						uint64_t o = p->sum_len + j;
+						int c = seq_nt4_table[(uint8_t)s->seq[i].seq[j]];
+						mm_seq4_set(p->mi->S, o, c);
+					}
 				}
 				// update p->sum_len and p->mi->n_seq
 				p->sum_len += seq->len;
@@ -269,7 +319,10 @@ static void *worker_pipeline(void *shared, int step, void *in)
         step_t *s = (step_t*)in;
 		for (i = 0; i < s->n_seq; ++i) {
 			mm_bseq1_t *t = &s->seq[i];
-			mm_sketch(0, t->seq, t->l_seq, p->mi->w, p->mi->k, t->rid, p->mi->is_hpc, &s->a);
+			if (t->l_seq > 0)
+				mm_sketch(0, t->seq, t->l_seq, p->mi->w, p->mi->k, t->rid, p->mi->flag&MM_I_HPC, &s->a);
+			else if (mm_verbose >= 2)
+				fprintf(stderr, "[WARNING] the length database sequence '%s' is 0\n", t->name);
 			free(t->seq); free(t->name);
 		}
 		free(s->seq); s->seq = 0;
@@ -277,21 +330,20 @@ static void *worker_pipeline(void *shared, int step, void *in)
     } else if (step == 2) { // dispatch sketch to buckets
         step_t *s = (step_t*)in;
 		mm_idx_add(p->mi, s->a.n, s->a.a);
-		free(s->a.a); free(s);
+		kfree(0, s->a.a); free(s);
 	}
     return 0;
 }
 
-mm_idx_t *mm_idx_gen(mm_bseq_file_t *fp, int w, int k, int b, int is_hpc, int mini_batch_size, int n_threads, uint64_t batch_size, int keep_name)
+mm_idx_t *mm_idx_gen(mm_bseq_file_t *fp, int w, int k, int b, int flag, int mini_batch_size, int n_threads, uint64_t batch_size)
 {
 	pipeline_t pl;
 	if (fp == 0 || mm_bseq_eof(fp)) return 0;
 	memset(&pl, 0, sizeof(pipeline_t));
-	pl.mini_batch_size = mini_batch_size < batch_size? mini_batch_size : batch_size;
-	pl.keep_name = keep_name;
+	pl.mini_batch_size = (uint64_t)mini_batch_size < batch_size? mini_batch_size : batch_size;
 	pl.batch_size = batch_size;
 	pl.fp = fp;
-	pl.mi = mm_idx_init(w, k, b, is_hpc);
+	pl.mi = mm_idx_init(w, k, b, flag);
 
 	kt_pipeline(n_threads < 3? n_threads : 3, worker_pipeline, &pl, 3);
 	if (mm_verbose >= 3)
@@ -304,17 +356,66 @@ mm_idx_t *mm_idx_gen(mm_bseq_file_t *fp, int w, int k, int b, int is_hpc, int mi
 	return pl.mi;
 }
 
-mm_idx_t *mm_idx_build(const char *fn, int w, int k, int is_hpc, int n_threads) // a simpler interface
+mm_idx_t *mm_idx_build(const char *fn, int w, int k, int flag, int n_threads) // a simpler interface; deprecated
 {
 	mm_bseq_file_t *fp;
 	mm_idx_t *mi;
 	fp = mm_bseq_open(fn);
 	if (fp == 0) return 0;
-	mi = mm_idx_gen(fp, w, k, MM_IDX_DEF_B, is_hpc, 1<<18, n_threads, UINT64_MAX, 1);
+	mi = mm_idx_gen(fp, w, k, 14, flag, 1<<18, n_threads, UINT64_MAX);
 	mm_bseq_close(fp);
 	return mi;
 }
 
+mm_idx_t *mm_idx_str(int w, int k, int is_hpc, int bucket_bits, int n, const char **seq, const char **name)
+{
+	uint64_t sum_len = 0;
+	mm128_v a = {0,0,0};
+	mm_idx_t *mi;
+	khash_t(str) *h;
+	int i, flag = 0;
+
+	if (n <= 0) return 0;
+	for (i = 0; i < n; ++i) // get the total length
+		sum_len += strlen(seq[i]);
+	if (is_hpc) flag |= MM_I_HPC;
+	if (name == 0) flag |= MM_I_NO_NAME;
+	if (bucket_bits < 0) bucket_bits = 14;
+	mi = mm_idx_init(w, k, bucket_bits, flag);
+	mi->n_seq = n;
+	mi->seq = (mm_idx_seq_t*)kcalloc(mi->km, n, sizeof(mm_idx_seq_t)); // ->seq is allocated from km
+	mi->S = (uint32_t*)calloc((sum_len + 7) / 8, 4);
+	mi->h = h = kh_init(str);
+	for (i = 0, sum_len = 0; i < n; ++i) {
+		const char *s = seq[i];
+		mm_idx_seq_t *p = &mi->seq[i];
+		uint32_t j;
+		if (name && name[i]) {
+			int absent;
+			p->name = (char*)kmalloc(mi->km, strlen(name[i]) + 1);
+			strcpy(p->name, name[i]);
+			kh_put(str, h, p->name, &absent);
+			assert(absent);
+		}
+		p->offset = sum_len;
+		p->len = strlen(s);
+		for (j = 0; j < p->len; ++j) {
+			int c = seq_nt4_table[(uint8_t)s[j]];
+			uint64_t o = sum_len + j;
+			mm_seq4_set(mi->S, o, c);
+		}
+		sum_len += p->len;
+		if (p->len > 0) {
+			a.n = 0;
+			mm_sketch(0, s, p->len, w, k, i, is_hpc, &a);
+			mm_idx_add(mi, a.n, a.a);
+		}
+	}
+	free(a.a);
+	mm_idx_post(mi, 1);
+	return mi;
+}
+
 /*************
  * index I/O *
  *************/
@@ -322,17 +423,20 @@ mm_idx_t *mm_idx_build(const char *fn, int w, int k, int is_hpc, int n_threads)
 void mm_idx_dump(FILE *fp, const mm_idx_t *mi)
 {
 	uint64_t sum_len = 0;
-	uint32_t x[5];
-	int i;
+	uint32_t x[5], i;
 
-	x[0] = mi->w, x[1] = mi->k, x[2] = mi->b, x[3] = mi->n_seq, x[4] = mi->is_hpc;
+	x[0] = mi->w, x[1] = mi->k, x[2] = mi->b, x[3] = mi->n_seq, x[4] = mi->flag;
 	fwrite(MM_IDX_MAGIC, 1, 4, fp);
 	fwrite(x, 4, 5, fp);
 	for (i = 0; i < mi->n_seq; ++i) {
-		uint8_t l;
-		l = strlen(mi->seq[i].name);
-		fwrite(&l, 1, 1, fp);
-		fwrite(mi->seq[i].name, 1, l, fp);
+		if (mi->seq[i].name) {
+			uint8_t l = strlen(mi->seq[i].name);
+			fwrite(&l, 1, 1, fp);
+			fwrite(mi->seq[i].name, 1, l, fp);
+		} else {
+			uint8_t l = 0;
+			fwrite(&l, 1, 1, fp);
+		}
 		fwrite(&mi->seq[i].len, 4, 1, fp);
 		sum_len += mi->seq[i].len;
 	}
@@ -352,15 +456,15 @@ void mm_idx_dump(FILE *fp, const mm_idx_t *mi)
 			fwrite(x, 8, 2, fp);
 		}
 	}
-	fwrite(mi->S, 4, (sum_len + 7) / 8, fp);
+	if (!(mi->flag & MM_I_NO_SEQ))
+		fwrite(mi->S, 4, (sum_len + 7) / 8, fp);
 	fflush(fp);
 }
 
 mm_idx_t *mm_idx_load(FILE *fp)
 {
-	int i;
 	char magic[4];
-	uint32_t x[5];
+	uint32_t x[5], i;
 	uint64_t sum_len = 0;
 	mm_idx_t *mi;
 
@@ -374,9 +478,11 @@ mm_idx_t *mm_idx_load(FILE *fp)
 		uint8_t l;
 		mm_idx_seq_t *s = &mi->seq[i];
 		fread(&l, 1, 1, fp);
-		s->name = (char*)kmalloc(mi->km, l + 1);
-		fread(s->name, 1, l, fp);
-		s->name[l] = 0;
+		if (l) {
+			s->name = (char*)kmalloc(mi->km, l + 1);
+			fread(s->name, 1, l, fp);
+			s->name[l] = 0;
+		}
 		fread(&s->len, 4, 1, fp);
 		s->offset = sum_len;
 		sum_len += s->len;
@@ -402,26 +508,80 @@ mm_idx_t *mm_idx_load(FILE *fp)
 			kh_val(h, k) = x[1];
 		}
 	}
-	mi->S = (uint32_t*)malloc((sum_len + 7) / 8 * 4);
-	fread(mi->S, 4, (sum_len + 7) / 8, fp);
+	if (!(mi->flag & MM_I_NO_SEQ)) {
+		mi->S = (uint32_t*)malloc((sum_len + 7) / 8 * 4);
+		fread(mi->S, 4, (sum_len + 7) / 8, fp);
+	}
 	return mi;
 }
 
-int mm_idx_is_idx(const char *fn)
+int64_t mm_idx_is_idx(const char *fn)
 {
 	int fd, is_idx = 0;
-	off_t ret;
+	int64_t ret, off_end;
 	char magic[4];
 
 	if (strcmp(fn, "-") == 0) return 0; // read from pipe; not an index
 	fd = open(fn, O_RDONLY);
 	if (fd < 0) return -1; // error
-	if ((ret = lseek(fd, 0, SEEK_END)) >= 4) {
+#ifdef WIN32
+	if ((off_end = _lseeki64(fd, 0, SEEK_END)) >= 4) {
+		_lseeki64(fd, 0, SEEK_SET);
+#else
+	if ((off_end = lseek(fd, 0, SEEK_END)) >= 4) {
 		lseek(fd, 0, SEEK_SET);
+#endif // WIN32
 		ret = read(fd, magic, 4);
 		if (ret == 4 && strncmp(magic, MM_IDX_MAGIC, 4) == 0)
 			is_idx = 1;
 	}
 	close(fd);
-	return is_idx;
+	return is_idx? off_end : 0;
+}
+
+mm_idx_reader_t *mm_idx_reader_open(const char *fn, const mm_idxopt_t *opt, const char *fn_out)
+{
+	int64_t is_idx;
+	mm_idx_reader_t *r;
+	is_idx = mm_idx_is_idx(fn);
+	if (is_idx < 0) return 0; // failed to open the index
+	r = (mm_idx_reader_t*)calloc(1, sizeof(mm_idx_reader_t));
+	r->is_idx = is_idx;
+	if (opt) r->opt = *opt;
+	else mm_idxopt_init(&r->opt);
+	if (r->is_idx) {
+		r->fp.idx = fopen(fn, "rb");
+		r->idx_size = is_idx;
+	} else r->fp.seq = mm_bseq_open(fn);
+	if (fn_out) r->fp_out = fopen(fn_out, "wb");
+	return r;
+}
+
+void mm_idx_reader_close(mm_idx_reader_t *r)
+{
+	if (r->is_idx) fclose(r->fp.idx);
+	else mm_bseq_close(r->fp.seq);
+	if (r->fp_out) fclose(r->fp_out);
+	free(r);
+}
+
+mm_idx_t *mm_idx_reader_read(mm_idx_reader_t *r, int n_threads)
+{
+	mm_idx_t *mi;
+	if (r->is_idx) {
+		mi = mm_idx_load(r->fp.idx);
+		if (mi && mm_verbose >= 2 && (mi->k != r->opt.k || mi->w != r->opt.w || (mi->flag&MM_I_HPC) != (r->opt.flag&MM_I_HPC)))
+			fprintf(stderr, "[WARNING]\033[1;31m Indexing parameters (-k, -w or -H) overridden by parameters used in the prebuilt index.\033[0m\n");
+	} else
+		mi = mm_idx_gen(r->fp.seq, r->opt.w, r->opt.k, r->opt.bucket_bits, r->opt.flag, r->opt.mini_batch_size, n_threads, r->opt.batch_size);
+	if (mi) {
+		if (r->fp_out) mm_idx_dump(r->fp_out, mi);
+		mi->index = r->n_parts++;
+	}
+	return mi;
+}
+
+int mm_idx_reader_eof(const mm_idx_reader_t *r) // TODO: in extremely rare cases, mm_bseq_eof() might not work
+{
+	return r->is_idx? (feof(r->fp.idx) || ftell(r->fp.idx) == r->idx_size) : mm_bseq_eof(r->fp.seq);
 }

kalloc.c

@@ -1,175 +1,144 @@
 #include <stdio.h>
 #include <stdlib.h>
 #include <string.h>
-#include <limits.h>
 #include "kalloc.h"
 
-/* The whole thing is: ("@" for the kheader_t of the block, "-" for free
- * memory, and "+" for allocated memory. One char for one unit.)
- *                        
- *           This region is core 1.                             This region is core 2.
+/* In kalloc, a *core* is a large chunk of contiguous memory. Each core is
+ * associated with a master header, which keeps the size of the current core
+ * and the pointer to next core. Kalloc allocates small *blocks* of memory from
+ * the cores and organizes free memory blocks in a circular single-linked list.
  *
- *   @-------@++++++@++++++++++++@------------           @----------@++++++++++++@+++++++@------------
- *   |                           |                       |                               |
- *   p=p->ptr->ptr->ptr->ptr     p->ptr             p->ptr->ptr                p->ptr->ptr->ptr
+ * In the following diagram, "@" stands for the header of a free block (of type
+ * header_t), "#" for the header of an allocated block (of type size_t), "-"
+ * for free memory, and "+" for allocated memory.
+ *
+ * master        This region is core 1.          master           This region is core 2.
+ *      |                                             |
+ *      *@-------#++++++#++++++++++++@--------        *@----------#++++++++++++#+++++++@------------
+ *       |                           |                 |                               |
+ *       p=p->ptr->ptr->ptr->ptr     p->ptr            p->ptr->ptr                     p->ptr->ptr->ptr
  */
 
-#define PTR(p) ((size_t*)((size_t*)p)[1])
+#define MIN_CORE_SIZE 0x80000
 
-typedef struct _allocated_t {
-	struct _allocated_t *next;
-	size_t *ptr;
-} allocated_t;
+typedef struct header_t {
+	size_t size;
+	struct header_t *ptr;
+} header_t;
 
 typedef struct {
-	size_t base[2], *loop_head;
-	allocated_t list_head, *list_tail;
-	size_t total_allocated;
+	header_t base, *loop_head, *core_head; /* base is a zero-sized block always kept in the loop */
 } kmem_t;
 
-void *km_init()
-{
-	return calloc(1, sizeof(kmem_t));
-}
-
-static void kerror(const char *s)
+static void panic(const char *s)
 {
 	fprintf(stderr, "%s\n", s);
-	exit(1);
+	abort();
 }
 
-static size_t *morecore(kmem_t *km, size_t nu)
+void *km_init(void)
 {
-	size_t rnu, *up;
-
-	rnu = (nu + 0xfffff) & (~(size_t)0xfffff);
-	up = (size_t*)malloc(rnu * sizeof(size_t));
-	if (!up) { /* fail to allocate memory */
-		km_stat(km);
-		fprintf(stderr, "[morecore] %lu bytes requested but not available.\n", rnu * sizeof(size_t));
-		exit(1);
-	}
-	/* put the pointer in km->list_head */
-	if (km->list_tail == 0) km->list_tail = &km->list_head;
-	km->list_tail->ptr = up;
-	km->list_tail->next = (allocated_t*)calloc(1, sizeof(allocated_t));
-	km->list_tail = km->list_tail->next;
-
-	km->total_allocated += rnu * sizeof(size_t);
-	*up = rnu; /* the size of the current block, and in this case the block is the same as the new core */
-	kfree(km, up + 1); /* initialize the new "core" */
-	return km->loop_head;
+	return calloc(1, sizeof(kmem_t));
 }
 
 void km_destroy(void *_km)
 {
 	kmem_t *km = (kmem_t*)_km;
-	allocated_t *p, *q;
-	if (km == 0) return;
-	p = &km->list_head;
-	do {
-		q = p->next;
-		free(p->ptr);
-		if (p != &km->list_head) free(p);
+	header_t *p, *q;
+	if (km == NULL) return;
+	for (p = km->core_head; p != NULL;) {
+		q = p->ptr;
+		free(p);
 		p = q;
-	} while (p && p->next);
-	if (p != &km->list_head) free(p);
+	}
 	free(km);
 }
 
-void kfree(void *_km, void *ap)
+static header_t *morecore(kmem_t *km, size_t nu)
 {
-	size_t *p, *q;
+	header_t *q;
+	size_t bytes, *p;
+	nu = (nu + 1 + (MIN_CORE_SIZE - 1)) / MIN_CORE_SIZE * MIN_CORE_SIZE; /* the first +1 for core header */
+	bytes = nu * sizeof(header_t);
+	q = (header_t*)malloc(bytes);
+	if (!q) panic("[morecore] insufficient memory");
+	q->ptr = km->core_head, q->size = nu, km->core_head = q;
+	p = (size_t*)(q + 1);
+	*p = nu - 1; /* the size of the free block; -1 because the first unit is used for the core header */
+	kfree(km, p + 1); /* initialize the new "core"; NB: the core header is not looped. */
+	return km->loop_head;
+}
+
+void kfree(void *_km, void *ap) /* kfree() also adds a new core to the circular list */
+{
+	header_t *p, *q;
 	kmem_t *km = (kmem_t*)_km;
 	
 	if (!ap) return;
-	if (km == 0) {
+	if (km == NULL) {
 		free(ap);
 		return;
 	}
-	p = (size_t*)ap - 1; /* *p is the size of the current block */
+	p = (header_t*)((size_t*)ap - 1);
+	p->size = *((size_t*)ap - 1);
 	/* Find the pointer that points to the block to be freed. The following loop can stop on two conditions:
 	 *
-	 * a) "p>q && p<q->ptr": @------@++++++++@+++++++@-------    @---------------@+++++++@-------
+	 * a) "p>q && p<q->ptr": @------#++++++++#+++++++@-------    @---------------#+++++++@-------
 	 *    (can also be in    |      |                |        -> |                       |
 	 *     two cores)        q      p           q->ptr           q                  q->ptr
 	 *
-	 *                       @--------    @+++++++++@--------    @--------    @------------------
+	 *                       @--------    #+++++++++@--------    @--------    @------------------
 	 *                       |            |         |         -> |            |
 	 *                       q            p    q->ptr            q       q->ptr
 	 *
-	 * b) "q>=q->ptr && (p>q || p<q->ptr)":  @-------@+++++   @--------@+++++++     @-------@+++++   @----------------
+	 * b) "q>=q->ptr && (p>q || p<q->ptr)":  @-------#+++++   @--------#+++++++     @-------#+++++   @----------------
 	 *                                       |                |        |         -> |                |
 	 *                                  q->ptr                q        p       q->ptr                q
 	 *
-	 *                                       @+++++++@-----   @++++++++@-------     @-------------   @++++++++@-------
+	 *                                       #+++++++@-----   #++++++++@-------     @-------------   #++++++++@-------
 	 *                                       |       |                 |         -> |                         |
 	 *                                       p  q->ptr                 q       q->ptr                         q
 	 */
-	for (q = km->loop_head; !(p > q && p < PTR(q)); q = PTR(q))
-		if (q >= PTR(q) && (p > q || p < PTR(q))) break;
-	if (p + (*p) == PTR(q)) { /* two adjacent blocks, merge p and q->ptr (the 2nd and 4th cases) */
-		*p += *PTR(q); /* this is the new q->ptr size */
-		p[1] = (size_t)PTR(PTR(q)); /* this is the new q->ptr->ptr */
-		/* p is actually the new q->ptr. The actual change happens a few lines below. */
-	} else if (p + (*p) > PTR(q) && PTR(q) >= p) { /* the end of the allocated block is in the next free block */
-		kerror("[kfree] The end of the allocated block enters a free block.");
-	} else p[1] = (size_t)PTR(q); /* backup q->ptr */
+	for (q = km->loop_head; !(p > q && p < q->ptr); q = q->ptr)
+		if (q >= q->ptr && (p > q || p < q->ptr)) break;
+	if (p + p->size == q->ptr) { /* two adjacent blocks, merge p and q->ptr (the 2nd and 4th cases) */
+		p->size += q->ptr->size;
+		p->ptr = q->ptr->ptr;
+	} else if (p + p->size > q->ptr && q->ptr >= p) {
+		panic("[kfree] The end of the allocated block enters a free block.");
+	} else p->ptr = q->ptr; /* backup q->ptr */
 
-	if (q + (*q) == p) { /* two adjacent blocks, merge q and p (the other two cases) */
-		*q += *p;
-		q[1] = (size_t)PTR(p);
+	if (q + q->size == p) { /* two adjacent blocks, merge q and p (the other two cases) */
+		q->size += p->size;
+		q->ptr = p->ptr;
 		km->loop_head = q;
-	} else if (q + (*q) > p && p >= q) { /* the end of a free block in the allocated block */
-		kerror("[kfree] The end of a free block enters the allocated block.");
-	} else km->loop_head = p, q[1] = (size_t)p; /* in two cores, cannot be merged */
-}
-
-void *krealloc(void *_km, void *ap, size_t n_bytes)
-{
-	kmem_t *km = (kmem_t*)_km;
-	size_t n_units, *p, *q;
-
-	if (n_bytes == 0) {
-		kfree(km, ap); return 0;
-	}
-	if (km == 0) return realloc(ap, n_bytes);
-	if (!ap) return kmalloc(km, n_bytes);
-	n_units = 1 + (n_bytes + sizeof(size_t) - 1) / sizeof(size_t);
-	p = (size_t*)ap - 1;
-	if (*p >= n_units) return ap; /* TODO: this prevents shrinking */
-	q = (size_t*)kmalloc(km, n_bytes);
-	memcpy(q, ap, (*p - 1) * sizeof(size_t));
-	kfree(km, ap);
-	return q;
+	} else if (q + q->size > p && p >= q) {
+		panic("[kfree] The end of a free block enters the allocated block.");
+	} else km->loop_head = p, q->ptr = p; /* in two cores, cannot be merged; create a new block in the list */
 }
 
 void *kmalloc(void *_km, size_t n_bytes)
 {
 	kmem_t *km = (kmem_t*)_km;
-	size_t n_units, *p, *q;
+	size_t n_units;
+	header_t *p, *q;
 
 	if (n_bytes == 0) return 0;
-	if (km == 0) return malloc(n_bytes);
-	/* "n_units" means the number of units. The size of one unit equals to sizeof(kheader_t).
-	 * "1" is the kheader_t of a block, which is always required. */
-	n_units = 1 + (n_bytes + sizeof(size_t) - 1) / sizeof(size_t);
-	if (n_units&1) ++n_units; /* make n_units an even number, or it will segfault if only one unit remains */
+	if (km == NULL) return malloc(n_bytes);
+	n_units = (n_bytes + sizeof(size_t) + sizeof(header_t) - 1) / sizeof(header_t) + 1;
 
-	if (!(q = km->loop_head)) { /* the first time when kmalloc() is called, intialization */
-		km->base[1] = (size_t)(km->loop_head = q = km->base); *q = 0;
-	}
-	for (p = PTR(q);; q = p, p = PTR(p)) { /* search for a suitable block */
-		if (*p >= n_units) { /* p->size if the size of current block. This line means the current block is large enough. */
-			if (*p == n_units) q[1] = (size_t)PTR(p); /* no need to split the block */
-			else { /* split the block */
-				/* memory is allocated at the end of the block */
-				*p -= n_units; /* reduce the size of the free block */
-				p += *p; /* skip to the kheader_t of the allocated block */
-				*p = n_units; /* set the size */
+	if (!(q = km->loop_head)) /* the first time when kmalloc() is called, intialize it */
+		q = km->loop_head = km->base.ptr = &km->base;
+	for (p = q->ptr;; q = p, p = p->ptr) { /* search for a suitable block */
+		if (p->size >= n_units) { /* p->size if the size of current block. This line means the current block is large enough. */
+			if (p->size == n_units) q->ptr = p->ptr; /* no need to split the block */
+			else { /* split the block. NB: memory is allocated at the end of the block! */
+				p->size -= n_units; /* reduce the size of the free block */
+				p += p->size; /* p points to the allocated block */
+				*(size_t*)p = n_units; /* set the size */
 			}
 			km->loop_head = q; /* set the end of chain */
-			return p + 1; /* skip the kheader_t */
+			return (size_t*)p + 1;
 		}
 		if (p == km->loop_head) { /* then ask for more "cores" */
 			if ((p = morecore(km, n_units)) == 0) return 0;
@@ -182,33 +151,48 @@ void *kcalloc(void *_km, size_t count, size_t size)
 	kmem_t *km = (kmem_t*)_km;
 	void *p;
 	if (size == 0 || count == 0) return 0;
-	if (km == 0) return calloc(count, size);
+	if (km == NULL) return calloc(count, size);
 	p = kmalloc(km, count * size);
 	memset(p, 0, count * size);
 	return p;
 }
 
-void km_stat(const void *_km)
+void *krealloc(void *_km, void *ap, size_t n_bytes) // TODO: this can be made more efficient in principle
 {
 	kmem_t *km = (kmem_t*)_km;
-	unsigned n_blocks, n_units;
-	size_t max_block = 0, *p, *q;
-	float frag;
+	size_t n_units, *p, *q;
 
-	if (km == 0 || !(p = km->loop_head)) return;
-	n_blocks = n_units = 0;
-	do {
-		q = PTR(p);
-		if (*p > max_block) max_block = *p;
-		n_units += *p;
-		if (p + (*p) > q && q > p)
-			kerror("[kr_stat] The end of a free block enters another free block.");
-		p = q;
-		++n_blocks;
-	} while (p != km->loop_head);
-	
-	--n_blocks;
-	frag = 1.0/1024.0 * n_units * sizeof(size_t) / n_blocks;
-	fprintf(stderr, "[kr_stat] tot=%lu, free=%lu, n_block=%u, max_block=%lu, frag_len=%.3fK\n",
-			km->total_allocated, n_units * sizeof(size_t), n_blocks, max_block * sizeof(size_t), frag);
+	if (n_bytes == 0) {
+		kfree(km, ap); return 0;
+	}
+	if (km == NULL) return realloc(ap, n_bytes);
+	if (ap == NULL) return kmalloc(km, n_bytes);
+	n_units = (n_bytes + sizeof(size_t) + sizeof(header_t) - 1) / sizeof(header_t);
+	p = (size_t*)ap - 1;
+	if (*p >= n_units) return ap; /* TODO: this prevents shrinking */
+	q = (size_t*)kmalloc(km, n_bytes);
+	memcpy(q, ap, (*p - 1) * sizeof(header_t));
+	kfree(km, ap);
+	return q;
+}
+
+void km_stat(const void *_km, km_stat_t *s)
+{
+	kmem_t *km = (kmem_t*)_km;
+	header_t *p;
+	memset(s, 0, sizeof(km_stat_t));
+	if (km == NULL || km->loop_head == NULL) return;
+	for (p = km->loop_head;; p = p->ptr) {
+		s->available += p->size * sizeof(header_t);
+		if (p->size != 0) ++s->n_blocks; /* &kmem_t::base is always one of the cores. It is zero-sized. */
+		if (p->ptr > p && p + p->size > p->ptr)
+			panic("[km_stat] The end of a free block enters another free block.");
+		if (p->ptr == km->loop_head) break;
+	}
+	for (p = km->core_head; p != NULL; p = p->ptr) {
+		size_t size = p->size * sizeof(header_t);
+		++s->n_cores;
+		s->capacity += size;
+		s->largest = s->largest > size? s->largest : size;
+	}
 }

kalloc.h

@@ -1,14 +1,16 @@
 #ifndef _KALLOC_H_
 #define _KALLOC_H_
 
-#include <stdlib.h>
-
-#define km_size(x) (*(((size_t*)(x))-1) * sizeof(size_t))
+#include <stddef.h> /* for size_t */
 
 #ifdef __cplusplus
 extern "C" {
 #endif
 
+typedef struct {
+	size_t capacity, available, n_blocks, n_cores, largest;
+} km_stat_t;
+
 void *kmalloc(void *km, size_t size);
 void *krealloc(void *km, void *ptr, size_t size);
 void *kcalloc(void *km, size_t count, size_t size);
@@ -16,8 +18,7 @@ void kfree(void *km, void *ptr);
 
 void *km_init(void);
 void km_destroy(void *km);
-
-void km_stat(const void *km); // TODO: return numbers instead of print to stderr
+void km_stat(const void *_km, km_stat_t *s);
 
 #ifdef __cplusplus
 }

kdq.h

@@ -3,11 +3,12 @@
 
 #include <stdlib.h>
 #include <string.h>
+#include <stdint.h>
 #include "kalloc.h"
 
 #define __KDQ_TYPE(type) \
 	typedef struct { \
-		size_t front:58, bits:6, count, mask; \
+		uint64_t front:58, bits:6, count, mask; \
 		type *a; \
 		void *km; \
 	} kdq_##type##_t;

ketopt.h

@@ -0,0 +1,116 @@
+#ifndef KETOPT_H
+#define KETOPT_H
+
+#include <string.h> /* for strchr() and strncmp() */
+
+#define ko_no_argument       0
+#define ko_required_argument 1
+#define ko_optional_argument 2
+
+typedef struct {
+	int ind;   /* equivalent to optind */
+	int opt;   /* equivalent to optopt */
+	char *arg; /* equivalent to optarg */
+	int longidx; /* index of a long option; or -1 if short */
+	/* private variables not intended for external uses */
+	int i, pos, n_args;
+} ketopt_t;
+
+typedef struct {
+	char *name;
+	int has_arg;
+	int val;
+} ko_longopt_t;
+
+static ketopt_t KETOPT_INIT = { 1, 0, 0, -1, 1, 0, 0 };
+
+static void ketopt_permute(char *argv[], int j, int n) /* move argv[j] over n elements to the left */
+{
+	int k;
+	char *p = argv[j];
+	for (k = 0; k < n; ++k)
+		argv[j - k] = argv[j - k - 1];
+	argv[j - k] = p;
+}
+
+/**
+ * Parse command-line options and arguments
+ *
+ * This fuction has a similar interface to GNU's getopt_long(). Each call
+ * parses one option and returns the option name.  s->arg points to the option
+ * argument if present. The function returns -1 when all command-line arguments
+ * are parsed. In this case, s->ind is the index of the first non-option
+ * argument.
+ *
+ * @param s         status; shall be initialized to KETOPT_INIT on the first call
+ * @param argc      length of argv[]
+ * @param argv      list of command-line arguments; argv[0] is ignored
+ * @param permute   non-zero to move options ahead of non-option arguments
+ * @param ostr      option string
+ * @param longopts  long options
+ *
+ * @return ASCII for a short option; ko_longopt_t::val for a long option; -1 if
+ *         argv[] is fully processed; '?' for an unknown option or an ambiguous
+ *         long option; ':' if an option argument is missing
+ */
+static int ketopt(ketopt_t *s, int argc, char *argv[], int permute, const char *ostr, const ko_longopt_t *longopts)
+{
+	int opt = -1, i0, j;
+	if (permute) {
+		while (s->i < argc && (argv[s->i][0] != '-' || argv[s->i][1] == '\0'))
+			++s->i, ++s->n_args;
+	}
+	s->arg = 0, s->longidx = -1, i0 = s->i;
+	if (s->i >= argc || argv[s->i][0] != '-' || argv[s->i][1] == '\0') {
+		s->ind = s->i - s->n_args;
+		return -1;
+	}
+	if (argv[s->i][0] == '-' && argv[s->i][1] == '-') { /* "--" or a long option */
+		if (argv[s->i][2] == '\0') { /* a bare "--" */
+			ketopt_permute(argv, s->i, s->n_args);
+			++s->i, s->ind = s->i - s->n_args;
+			return -1;
+		}
+		s->opt = 0, opt = '?', s->pos = -1;
+		if (longopts) { /* parse long options */
+			int k, n_matches = 0;
+			const ko_longopt_t *o = 0;
+			for (j = 2; argv[s->i][j] != '\0' && argv[s->i][j] != '='; ++j) {} /* find the end of the option name */
+			for (k = 0; longopts[k].name != 0; ++k)
+				if (strncmp(&argv[s->i][2], longopts[k].name, j - 2) == 0)
+					++n_matches, o = &longopts[k];
+			if (n_matches == 1) {
+				s->opt = opt = o->val, s->longidx = o - longopts;
+				if (argv[s->i][j] == '=') s->arg = &argv[s->i][j + 1];
+				if (o->has_arg == 1 && argv[s->i][j] == '\0') {
+					if (s->i < argc - 1) s->arg = argv[++s->i];
+					else opt = ':'; /* missing option argument */
+				}
+			}
+		}
+	} else { /* a short option */
+		char *p;
+		if (s->pos == 0) s->pos = 1;
+		opt = s->opt = argv[s->i][s->pos++];
+		p = strchr(ostr, opt);
+		if (p == 0) {
+			opt = '?'; /* unknown option */
+		} else if (p[1] == ':') {
+			if (argv[s->i][s->pos] == 0) {
+				if (s->i < argc - 1) s->arg = argv[++s->i];
+				else opt = ':'; /* missing option argument */
+			} else s->arg = &argv[s->i][s->pos];
+			s->pos = -1;
+		}
+	}
+	if (s->pos < 0 || argv[s->i][s->pos] == 0) {
+		++s->i, s->pos = 0;
+		if (s->n_args > 0) /* permute */
+			for (j = i0; j < s->i; ++j)
+				ketopt_permute(argv, j, s->n_args);
+	}
+	s->ind = s->i - s->n_args;
+	return opt;
+}
+
+#endif

ksort.h

@@ -30,6 +30,7 @@
 
 #include <stdlib.h>
 #include <string.h>
+#include <assert.h>
 
 typedef struct {
 	void *left, *right;
@@ -38,7 +39,24 @@ typedef struct {
 
 #define KSORT_SWAP(type_t, a, b) { register type_t t=(a); (a)=(b); (b)=t; }
 
-#define KSORT_INIT(name, type_t, __sort_lt)								\
+#define KSORT_INIT(name, type_t, __sort_lt) \
+	void ks_heapdown_##name(size_t i, size_t n, type_t l[]) \
+	{ \
+		size_t k = i; \
+		type_t tmp = l[i]; \
+		while ((k = (k << 1) + 1) < n) { \
+			if (k != n - 1 && __sort_lt(l[k], l[k+1])) ++k; \
+			if (__sort_lt(l[k], tmp)) break; \
+			l[i] = l[k]; i = k; \
+		} \
+		l[i] = tmp; \
+	} \
+	void ks_heapmake_##name(size_t lsize, type_t l[]) \
+	{ \
+		size_t i; \
+		for (i = (lsize >> 1) - 1; i != (size_t)(-1); --i) \
+			ks_heapdown_##name(i, lsize, l); \
+	} \
 	type_t ks_ksmall_##name(size_t n, type_t arr[], size_t kk)			\
 	{																	\
 		type_t *low, *high, *k, *ll, *hh, *mid;							\
@@ -78,6 +96,7 @@ typedef const char *ksstr_t;
 #define KSORT_INIT_STR KSORT_INIT(str, ksstr_t, ks_lt_str)
 
 #define RS_MIN_SIZE 64
+#define RS_MAX_BITS 8
 
 #define KRADIX_SORT_INIT(name, rstype_t, rskey, sizeof_key) \
 	typedef struct { \
@@ -98,7 +117,8 @@ typedef const char *ksstr_t;
 	{ \
 		rstype_t *i; \
 		int size = 1<<n_bits, m = size - 1; \
-		rsbucket_##name##_t *k, b[size], *be = b + size; \
+		rsbucket_##name##_t *k, b[1<<RS_MAX_BITS], *be = b + size; \
+		assert(n_bits <= RS_MAX_BITS); \
 		for (k = b; k != be; ++k) k->b = k->e = beg; \
 		for (i = beg; i != end; ++i) ++b[rskey(*i)>>s&m].e; \
 		for (k = b + 1; k != be; ++k) \
@@ -127,7 +147,7 @@ typedef const char *ksstr_t;
 	void radix_sort_##name(rstype_t *beg, rstype_t *end) \
 	{ \
 		if (end - beg <= RS_MIN_SIZE) rs_insertsort_##name(beg, end); \
-		else rs_sort_##name(beg, end, 8, sizeof_key * 8 - 8); \
+		else rs_sort_##name(beg, end, RS_MAX_BITS, (sizeof_key - 1) * RS_MAX_BITS); \
 	}
 
 #endif

ksw2.h

@@ -14,6 +14,7 @@
 #define KSW_EZ_REV_CIGAR   0x80 // reverse CIGAR in the output
 #define KSW_EZ_SPLICE_FOR  0x100
 #define KSW_EZ_SPLICE_REV  0x200
+#define KSW_EZ_SPLICE_FLANK 0x400
 
 #ifdef __cplusplus
 extern "C" {
@@ -26,6 +27,7 @@ typedef struct {
 	int mte, mte_q;        // max score when reaching the end of target
 	int score;             // max score reaching both ends; may be KSW_NEG_INF
 	int m_cigar, n_cigar;
+	int reach_end;
 	uint32_t *cigar;
 } ksw_extz_t;
 
@@ -46,14 +48,17 @@ typedef struct {
  * @param flag      flag (see KSW_EZ_* macros)
  * @param ez        (out) scores and cigar
  */
-void ksw_extz(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int flag, ksw_extz_t *ez);
-void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int flag, ksw_extz_t *ez);
+void ksw_extz(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+			  int8_t q, int8_t e, int w, int zdrop, int flag, ksw_extz_t *ez);
+
+void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez);
 
 void ksw_extd(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
 			  int8_t gapo, int8_t gape, int8_t gapo2, int8_t gape2, int w, int zdrop, int flag, ksw_extz_t *ez);
 
 void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
-				   int8_t gapo, int8_t gape, int8_t gapo2, int8_t gape2, int w, int zdrop, int flag, ksw_extz_t *ez);
+				   int8_t gapo, int8_t gape, int8_t gapo2, int8_t gape2, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez);
 
 void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
 				   int8_t gapo, int8_t gape, int8_t gapo2, int8_t noncan, int zdrop, int flag, ksw_extz_t *ez);
@@ -111,7 +116,7 @@ static inline uint32_t *ksw_push_cigar(void *km, int *n_cigar, int *m_cigar, uin
 //   bit 0-2: which type gets the max - 0 for H, 1 for E, 2 for F, 3 for \tilde{E} and 4 for \tilde{F}
 //   bit 3/0x08: 1 if a continuation on the E state (bit 5/0x20 for a continuation on \tilde{E})
 //   bit 4/0x10: 1 if a continuation on the F state (bit 6/0x40 for a continuation on \tilde{F})
-static inline void ksw_backtrack(void *km, int is_rot, int is_rev, int with_N, const uint8_t *p, const int *off, const int *off_end, int n_col, int i0, int j0,
+static inline void ksw_backtrack(void *km, int is_rot, int is_rev, int min_intron_len, const uint8_t *p, const int *off, const int *off_end, int n_col, int i0, int j0,
 								 int *m_cigar_, int *n_cigar_, uint32_t **cigar_)
 { // p[] - lower 3 bits: which type gets the max; bit
 	int n_cigar = 0, m_cigar = *m_cigar_, i = i0, j = j0, r, state = 0;
@@ -122,22 +127,22 @@ static inline void ksw_backtrack(void *km, int is_rot, int is_rev, int with_N, c
 			r = i + j;
 			if (i < off[r]) force_state = 2;
 			if (off_end && i > off_end[r]) force_state = 1;
-			tmp = force_state < 0? p[r * n_col + i - off[r]] : 0;
+			tmp = force_state < 0? p[(size_t)r * n_col + i - off[r]] : 0;
 		} else {
 			if (j < off[i]) force_state = 2;
 			if (off_end && j > off_end[i]) force_state = 1;
-			tmp = force_state < 0? p[i * n_col + j - off[i]] : 0;
+			tmp = force_state < 0? p[(size_t)i * n_col + j - off[i]] : 0;
 		}
 		if (state == 0) state = tmp & 7; // if requesting the H state, find state one maximizes it.
 		else if (!(tmp >> (state + 2) & 1)) state = 0; // if requesting other states, _state_ stays the same if it is a continuation; otherwise, set to H
 		if (state == 0) state = tmp & 7; // TODO: probably this line can be merged into the "else if" line right above; not 100% sure
 		if (force_state >= 0) state = force_state;
 		if (state == 0) cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, 0, 1), --i, --j; // match
-		else if (state == 1 || (state == 3 && !with_N)) cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, 2, 1), --i; // deletion
-		else if (state == 3 && with_N) cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, 3, 1), --i; // intron
+		else if (state == 1 || (state == 3 && min_intron_len <= 0)) cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, 2, 1), --i; // deletion
+		else if (state == 3 && min_intron_len > 0) cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, 3, 1), --i; // intron
 		else cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, 1, 1), --j; // insertion
 	}
-	if (i >= 0) cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, 2, i + 1); // first deletion
+	if (i >= 0) cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, min_intron_len > 0 && i >= min_intron_len? 3 : 2, i + 1); // first deletion
 	if (j >= 0) cigar = ksw_push_cigar(km, &n_cigar, &m_cigar, cigar, 1, j + 1); // first insertion
 	if (!is_rev)
 		for (i = 0; i < n_cigar>>1; ++i) // reverse CIGAR
@@ -149,7 +154,7 @@ static inline void ksw_reset_extz(ksw_extz_t *ez)
 {
 	ez->max_q = ez->max_t = ez->mqe_t = ez->mte_q = -1;
 	ez->max = 0, ez->score = ez->mqe = ez->mte = KSW_NEG_INF;
-	ez->n_cigar = 0, ez->zdropped = 0;
+	ez->n_cigar = 0, ez->zdropped = 0, ez->reach_end = 0;
 }
 
 static inline int ksw_apply_zdrop(ksw_extz_t *ez, int is_rot, int32_t H, int a, int b, int zdrop, int8_t e)
@@ -169,5 +174,4 @@ static inline int ksw_apply_zdrop(ksw_extz_t *ez, int is_rot, int32_t H, int a,
 	}
 	return 0;
 }
-
 #endif

ksw2_dispatch.c

@@ -0,0 +1,97 @@
+#ifdef KSW_CPU_DISPATCH
+#include <stdlib.h>
+#include "ksw2.h"
+
+#define SIMD_SSE     0x1
+#define SIMD_SSE2    0x2
+#define SIMD_SSE3    0x4
+#define SIMD_SSSE3   0x8
+#define SIMD_SSE4_1  0x10
+#define SIMD_SSE4_2  0x20
+#define SIMD_AVX     0x40
+#define SIMD_AVX2    0x80
+#define SIMD_AVX512F 0x100
+
+#ifndef _MSC_VER
+// adapted from https://github.com/01org/linux-sgx/blob/master/common/inc/internal/linux/cpuid_gnu.h
+void __cpuidex(int cpuid[4], int func_id, int subfunc_id)
+{
+#if defined(__x86_64__)
+	asm volatile ("cpuid"
+			: "=a" (cpuid[0]), "=b" (cpuid[1]), "=c" (cpuid[2]), "=d" (cpuid[3])
+			: "0" (func_id), "2" (subfunc_id));
+#else // on 32bit, ebx can NOT be used as PIC code
+	asm volatile ("xchgl %%ebx, %1; cpuid; xchgl %%ebx, %1"
+			: "=a" (cpuid[0]), "=r" (cpuid[1]), "=c" (cpuid[2]), "=d" (cpuid[3])
+			: "0" (func_id), "2" (subfunc_id));
+#endif
+}
+#endif
+
+int x86_simd(void)
+{
+	int flag = 0, cpuid[4], max_id;
+	__cpuidex(cpuid, 0, 0);
+	max_id = cpuid[0];
+	if (max_id == 0) return 0;
+	__cpuidex(cpuid, 1, 0);
+	if (cpuid[3]>>25&1) flag |= SIMD_SSE;
+	if (cpuid[3]>>26&1) flag |= SIMD_SSE2;
+	if (cpuid[2]>>0 &1) flag |= SIMD_SSE3;
+	if (cpuid[2]>>9 &1) flag |= SIMD_SSSE3;
+	if (cpuid[2]>>19&1) flag |= SIMD_SSE4_1;
+	if (cpuid[2]>>20&1) flag |= SIMD_SSE4_2;
+	if (cpuid[2]>>28&1) flag |= SIMD_AVX;
+	if (max_id >= 7) {
+		__cpuidex(cpuid, 7, 0);
+		if (cpuid[1]>>5 &1) flag |= SIMD_AVX2;
+		if (cpuid[1]>>16&1) flag |= SIMD_AVX512F;
+	}
+	return flag;
+}
+
+void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez)
+{
+	extern void ksw_extz2_sse2(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez);
+	extern void ksw_extz2_sse41(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez);
+	unsigned simd;
+	simd = x86_simd();
+	if (simd & SIMD_SSE4_1)
+		ksw_extz2_sse41(km, qlen, query, tlen, target, m, mat, q, e, w, zdrop, end_bonus, flag, ez);
+	else if (simd & SIMD_SSE2)
+		ksw_extz2_sse2(km, qlen, query, tlen, target, m, mat, q, e, w, zdrop, end_bonus, flag, ez);
+	else abort();
+}
+
+void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t e2, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez)
+{
+	extern void ksw_extd2_sse2(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t e2, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez);
+	extern void ksw_extd2_sse41(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t e2, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez);
+	unsigned simd;
+	simd = x86_simd();
+	if (simd & SIMD_SSE4_1)
+		ksw_extd2_sse41(km, qlen, query, tlen, target, m, mat, q, e, q2, e2, w, zdrop, end_bonus, flag, ez);
+	else if (simd & SIMD_SSE2)
+		ksw_extd2_sse2(km, qlen, query, tlen, target, m, mat, q, e, q2, e2, w, zdrop, end_bonus, flag, ez);
+	else abort();
+}
+
+void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t noncan, int zdrop, int flag, ksw_extz_t *ez)
+{
+	extern void ksw_exts2_sse2(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t noncan, int zdrop, int flag, ksw_extz_t *ez);
+	extern void ksw_exts2_sse41(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t noncan, int zdrop, int flag, ksw_extz_t *ez);
+	unsigned simd;
+	simd = x86_simd();
+	if (simd & SIMD_SSE4_1)
+		ksw_exts2_sse41(km, qlen, query, tlen, target, m, mat, q, e, q2, noncan, zdrop, flag, ez);
+	else if (simd & SIMD_SSE2)
+		ksw_exts2_sse2(km, qlen, query, tlen, target, m, mat, q, e, q2, noncan, zdrop, flag, ez);
+	else abort();
+}
+#endif

ksw2_extd2_sse.c

@@ -6,12 +6,26 @@
 #ifdef __SSE2__
 #include <emmintrin.h>
 
+#ifdef KSW_SSE2_ONLY
+#undef __SSE4_1__
+#endif
+
 #ifdef __SSE4_1__
 #include <smmintrin.h>
 #endif
 
+#ifdef KSW_CPU_DISPATCH
+#ifdef __SSE4_1__
+void ksw_extd2_sse41(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t e2, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez)
+#else
+void ksw_extd2_sse2(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t e2, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez)
+#endif
+#else
 void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
-				   int8_t q, int8_t e, int8_t q2, int8_t e2, int w, int zdrop, int flag, ksw_extz_t *ez)
+				   int8_t q, int8_t e, int8_t q2, int8_t e2, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez)
+#endif // ~KSW_CPU_DISPATCH
 {
 #define __dp_code_block1 \
 	z = _mm_load_si128(&s[t]); \
@@ -47,7 +61,7 @@ void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	int with_cigar = !(flag&KSW_EZ_SCORE_ONLY), approx_max = !!(flag&KSW_EZ_APPROX_MAX);
 	int32_t *H = 0, H0 = 0, last_H0_t = 0;
 	uint8_t *qr, *sf, *mem, *mem2 = 0;
-	__m128i q_, q2_, qe_, qe2_, zero_, sc_mch_, sc_mis_, m1_;
+	__m128i q_, q2_, qe_, qe2_, zero_, sc_mch_, sc_mis_, m1_, sc_N_;
 	__m128i *u, *v, *x, *y, *x2, *y2, *s, *p = 0;
 
 	ksw_reset_extz(ez);
@@ -62,6 +76,7 @@ void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	qe2_    = _mm_set1_epi8(q2 + e2);
 	sc_mch_ = _mm_set1_epi8(mat[0]);
 	sc_mis_ = _mm_set1_epi8(mat[1]);
+	sc_N_   = mat[m*m-1] == 0? _mm_set1_epi8(-e2) : _mm_set1_epi8(mat[m*m-1]);
 	m1_     = _mm_set1_epi8(m - 1); // wildcard
 
 	if (w < 0) w = tlen > qlen? tlen : qlen;
@@ -96,7 +111,7 @@ void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 		for (t = 0; t < tlen_ * 16; ++t) H[t] = KSW_NEG_INF;
 	}
 	if (with_cigar) {
-		mem2 = (uint8_t*)kmalloc(km, ((qlen + tlen - 1) * n_col_ + 1) * 16);
+		mem2 = (uint8_t*)kmalloc(km, ((size_t)(qlen + tlen - 1) * n_col_ + 1) * 16);
 		p = (__m128i*)(((size_t)mem2 + 15) >> 4 << 4);
 		off = (int*)kmalloc(km, (qlen + tlen - 1) * sizeof(int) * 2);
 		off_end = off + qlen + tlen - 1;
@@ -148,10 +163,11 @@ void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 				tmp = _mm_cmpeq_epi8(sq, st);
 #ifdef __SSE4_1__
 				tmp = _mm_blendv_epi8(sc_mis_, sc_mch_, tmp);
+				tmp = _mm_blendv_epi8(tmp,     sc_N_,   mask);
 #else
-				tmp = _mm_or_si128(_mm_andnot_si128(tmp, sc_mis_), _mm_and_si128(tmp, sc_mch_));
+				tmp = _mm_or_si128(_mm_andnot_si128(tmp,  sc_mis_), _mm_and_si128(tmp,  sc_mch_));
+				tmp = _mm_or_si128(_mm_andnot_si128(mask, tmp),     _mm_and_si128(mask, sc_N_));
 #endif
-				tmp = _mm_andnot_si128(mask, tmp);
 				_mm_storeu_si128((__m128i*)((int8_t*)s + t), tmp);
 			}
 		} else {
@@ -202,7 +218,7 @@ void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 #endif
 			}
 		} else if (!(flag&KSW_EZ_RIGHT)) { // gap left-alignment
-			__m128i *pr = p + r * n_col_ - st_;
+			__m128i *pr = p + (size_t)r * n_col_ - st_;
 			off[r] = st, off_end[r] = en;
 			for (t = st_; t <= en_; ++t) {
 				__m128i d, z, a, b, a2, b2, xt1, x2t1, vt1, ut, tmp;
@@ -249,7 +265,7 @@ void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 				_mm_store_si128(&pr[t], d);
 			}
 		} else { // gap right-alignment
-			__m128i *pr = p + r * n_col_ - st_;
+			__m128i *pr = p + (size_t)r * n_col_ - st_;
 			off[r] = st, off_end[r] = en;
 			for (t = st_; t <= en_; ++t) {
 				__m128i d, z, a, b, a2, b2, xt1, x2t1, vt1, ut, tmp;
@@ -364,10 +380,14 @@ void ksw_extd2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	if (!approx_max) kfree(km, H);
 	if (with_cigar) { // backtrack
 		int rev_cigar = !!(flag & KSW_EZ_REV_CIGAR);
-		if (!ez->zdropped && !(flag&KSW_EZ_EXTZ_ONLY))
+		if (!ez->zdropped && !(flag&KSW_EZ_EXTZ_ONLY)) {
 			ksw_backtrack(km, 1, rev_cigar, 0, (uint8_t*)p, off, off_end, n_col_*16, tlen-1, qlen-1, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
-		else if (ez->max_t >= 0 && ez->max_q >= 0)
+		} else if (!ez->zdropped && (flag&KSW_EZ_EXTZ_ONLY) && ez->mqe + end_bonus > (int)ez->max) {
+			ez->reach_end = 1;
+			ksw_backtrack(km, 1, rev_cigar, 0, (uint8_t*)p, off, off_end, n_col_*16, ez->mqe_t, qlen-1, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
+		} else if (ez->max_t >= 0 && ez->max_q >= 0) {
 			ksw_backtrack(km, 1, rev_cigar, 0, (uint8_t*)p, off, off_end, n_col_*16, ez->max_t, ez->max_q, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
+		}
 		kfree(km, mem2); kfree(km, off);
 	}
 }

ksw2_exts2_sse.c

@@ -6,12 +6,26 @@
 #ifdef __SSE2__
 #include <emmintrin.h>
 
+#ifdef KSW_SSE2_ONLY
+#undef __SSE4_1__
+#endif
+
 #ifdef __SSE4_1__
 #include <smmintrin.h>
 #endif
 
+#ifdef KSW_CPU_DISPATCH
+#ifdef __SSE4_1__
+void ksw_exts2_sse41(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t noncan, int zdrop, int flag, ksw_extz_t *ez)
+#else
+void ksw_exts2_sse2(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
+				   int8_t q, int8_t e, int8_t q2, int8_t noncan, int zdrop, int flag, ksw_extz_t *ez)
+#endif
+#else
 void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat,
 				   int8_t q, int8_t e, int8_t q2, int8_t noncan, int zdrop, int flag, ksw_extz_t *ez)
+#endif // ~KSW_CPU_DISPATCH
 {
 #define __dp_code_block1 \
 	z = _mm_load_si128(&s[t]); \
@@ -45,7 +59,7 @@ void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	int with_cigar = !(flag&KSW_EZ_SCORE_ONLY), approx_max = !!(flag&KSW_EZ_APPROX_MAX);
 	int32_t *H = 0, H0 = 0, last_H0_t = 0;
 	uint8_t *qr, *sf, *mem, *mem2 = 0;
-	__m128i q_, q2_, qe_, zero_, sc_mch_, sc_mis_, m1_;
+	__m128i q_, q2_, qe_, zero_, sc_mch_, sc_mis_, sc_N_, m1_;
 	__m128i *u, *v, *x, *y, *x2, *s, *p = 0, *donor, *acceptor;
 
 	ksw_reset_extz(ez);
@@ -57,6 +71,7 @@ void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	qe_     = _mm_set1_epi8(q + e);
 	sc_mch_ = _mm_set1_epi8(mat[0]);
 	sc_mis_ = _mm_set1_epi8(mat[1]);
+	sc_N_   = mat[m*m-1] == 0? _mm_set1_epi8(-e) : _mm_set1_epi8(mat[m*m-1]);
 	m1_     = _mm_set1_epi8(m - 1); // wildcard
 
 	tlen_ = (tlen + 15) / 16;
@@ -85,7 +100,7 @@ void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 		for (t = 0; t < tlen_ * 16; ++t) H[t] = KSW_NEG_INF;
 	}
 	if (with_cigar) {
-		mem2 = (uint8_t*)kmalloc(km, ((qlen + tlen - 1) * n_col_ + 1) * 16);
+		mem2 = (uint8_t*)kmalloc(km, ((size_t)(qlen + tlen - 1) * n_col_ + 1) * 16);
 		p = (__m128i*)(((size_t)mem2 + 15) >> 4 << 4);
 		off = (int*)kmalloc(km, (qlen + tlen - 1) * sizeof(int) * 2);
 		off_end = off + qlen + tlen - 1;
@@ -96,19 +111,22 @@ void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 
 	// set the donor and acceptor arrays. TODO: this assumes 0/1/2/3 encoding!
 	if (flag & (KSW_EZ_SPLICE_FOR|KSW_EZ_SPLICE_REV)) {
+		int semi_cost = flag&KSW_EZ_SPLICE_FLANK? -noncan/2 : 0; // GTr or yAG is worth 0.5 bit; see PMID:18688272
 		memset(donor, -noncan, tlen_ * 16);
-		for (t = 0; t < tlen - 2; ++t) {
-			int is_can = 0; // is a canonical site
-			if ((flag & KSW_EZ_SPLICE_FOR) && target[t+1] == 2 && target[t+2] == 3) is_can = 1;
-			if ((flag & KSW_EZ_SPLICE_REV) && target[t+1] == 1 && target[t+2] == 3) is_can = 1;
-			if (is_can) ((int8_t*)donor)[t] = 0;
+		for (t = 0; t < tlen - 4; ++t) {
+			int can_type = 0; // type of canonical site: 0=none, 1=GT/AG only, 2=GTr/yAG
+			if ((flag & KSW_EZ_SPLICE_FOR) && target[t+1] == 2 && target[t+2] == 3) can_type = 1; // GTr...
+			if ((flag & KSW_EZ_SPLICE_REV) && target[t+1] == 1 && target[t+2] == 3) can_type = 1; // CTr...
+			if (can_type && (target[t+3] == 0 || target[t+3] == 2)) can_type = 2;
+			if (can_type) ((int8_t*)donor)[t] = can_type == 2? 0 : semi_cost;
 		}
 		memset(acceptor, -noncan, tlen_ * 16);
 		for (t = 2; t < tlen; ++t) {
-			int is_can = 0;
-			if ((flag & KSW_EZ_SPLICE_FOR) && target[t-1] == 0 && target[t] == 2) is_can = 1;
-			if ((flag & KSW_EZ_SPLICE_REV) && target[t-1] == 0 && target[t] == 1) is_can = 1;
-			if (is_can) ((int8_t*)acceptor)[t] = 0;
+			int can_type = 0;
+			if ((flag & KSW_EZ_SPLICE_FOR) && target[t-1] == 0 && target[t] == 2) can_type = 1; // ...yAG
+			if ((flag & KSW_EZ_SPLICE_REV) && target[t-1] == 0 && target[t] == 1) can_type = 1; // ...yAC
+			if (can_type && (target[t-2] == 1 || target[t-2] == 3)) can_type = 2;
+			if (can_type) ((int8_t*)acceptor)[t] = can_type == 2? 0 : semi_cost;
 		}
 	}
 
@@ -145,10 +163,11 @@ void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 				tmp = _mm_cmpeq_epi8(sq, st);
 #ifdef __SSE4_1__
 				tmp = _mm_blendv_epi8(sc_mis_, sc_mch_, tmp);
+				tmp = _mm_blendv_epi8(tmp,     sc_N_,   mask);
 #else
-				tmp = _mm_or_si128(_mm_andnot_si128(tmp, sc_mis_), _mm_and_si128(tmp, sc_mch_));
+				tmp = _mm_or_si128(_mm_andnot_si128(tmp,  sc_mis_), _mm_and_si128(tmp,  sc_mch_));
+				tmp = _mm_or_si128(_mm_andnot_si128(mask, tmp),     _mm_and_si128(mask, sc_N_));
 #endif
-				tmp = _mm_andnot_si128(mask, tmp);
 				_mm_storeu_si128((__m128i*)((int8_t*)s + t), tmp);
 			}
 		} else {
@@ -348,9 +367,9 @@ void ksw_exts2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	if (with_cigar) { // backtrack
 		int rev_cigar = !!(flag & KSW_EZ_REV_CIGAR);
 		if (!ez->zdropped && !(flag&KSW_EZ_EXTZ_ONLY))
-			ksw_backtrack(km, 1, rev_cigar, 1, (uint8_t*)p, off, off_end, n_col_*16, tlen-1, qlen-1, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
+			ksw_backtrack(km, 1, rev_cigar, long_thres, (uint8_t*)p, off, off_end, n_col_*16, tlen-1, qlen-1, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
 		else if (ez->max_t >= 0 && ez->max_q >= 0)
-			ksw_backtrack(km, 1, rev_cigar, 1, (uint8_t*)p, off, off_end, n_col_*16, ez->max_t, ez->max_q, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
+			ksw_backtrack(km, 1, rev_cigar, long_thres, (uint8_t*)p, off, off_end, n_col_*16, ez->max_t, ez->max_q, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
 		kfree(km, mem2); kfree(km, off);
 	}
 }

ksw2_extz2_sse.c

@@ -5,11 +5,23 @@
 #ifdef __SSE2__
 #include <emmintrin.h>
 
+#ifdef KSW_SSE2_ONLY
+#undef __SSE4_1__
+#endif
+
 #ifdef __SSE4_1__
 #include <smmintrin.h>
 #endif
 
-void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int flag, ksw_extz_t *ez)
+#ifdef KSW_CPU_DISPATCH
+#ifdef __SSE4_1__
+void ksw_extz2_sse41(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez)
+#else
+void ksw_extz2_sse2(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez)
+#endif
+#else
+void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uint8_t *target, int8_t m, const int8_t *mat, int8_t q, int8_t e, int w, int zdrop, int end_bonus, int flag, ksw_extz_t *ez)
+#endif // ~KSW_CPU_DISPATCH
 {
 #define __dp_code_block1 \
 	z = _mm_add_epi8(_mm_load_si128(&s[t]), qe2_); \
@@ -38,7 +50,7 @@ void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	int with_cigar = !(flag&KSW_EZ_SCORE_ONLY), approx_max = !!(flag&KSW_EZ_APPROX_MAX);
 	int32_t *H = 0, H0 = 0, last_H0_t = 0;
 	uint8_t *qr, *sf, *mem, *mem2 = 0;
-	__m128i q_, qe2_, zero_, flag1_, flag2_, flag8_, flag16_, sc_mch_, sc_mis_, m1_, max_sc_;
+	__m128i q_, qe2_, zero_, flag1_, flag2_, flag8_, flag16_, sc_mch_, sc_mis_, sc_N_, m1_, max_sc_;
 	__m128i *u, *v, *x, *y, *s, *p = 0;
 
 	ksw_reset_extz(ez);
@@ -53,6 +65,7 @@ void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	flag16_ = _mm_set1_epi8(0x10);
 	sc_mch_ = _mm_set1_epi8(mat[0]);
 	sc_mis_ = _mm_set1_epi8(mat[1]);
+	sc_N_   = mat[m*m-1] == 0? _mm_set1_epi8(-e) : _mm_set1_epi8(mat[m*m-1]);
 	m1_     = _mm_set1_epi8(m - 1); // wildcard
 	max_sc_ = _mm_set1_epi8(mat[0] + (q + e) * 2);
 
@@ -76,7 +89,7 @@ void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 		for (t = 0; t < tlen_ * 16; ++t) H[t] = KSW_NEG_INF;
 	}
 	if (with_cigar) {
-		mem2 = (uint8_t*)kmalloc(km, ((qlen + tlen - 1) * n_col_ + 1) * 16);
+		mem2 = (uint8_t*)kmalloc(km, ((size_t)(qlen + tlen - 1) * n_col_ + 1) * 16);
 		p = (__m128i*)(((size_t)mem2 + 15) >> 4 << 4);
 		off = (int*)kmalloc(km, (qlen + tlen - 1) * sizeof(int) * 2);
 		off_end = off + qlen + tlen - 1;
@@ -118,10 +131,11 @@ void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 				tmp = _mm_cmpeq_epi8(sq, st);
 #ifdef __SSE4_1__
 				tmp = _mm_blendv_epi8(sc_mis_, sc_mch_, tmp);
+				tmp = _mm_blendv_epi8(tmp,     sc_N_,   mask);
 #else
-				tmp = _mm_or_si128(_mm_andnot_si128(tmp, sc_mis_), _mm_and_si128(tmp, sc_mch_));
+				tmp = _mm_or_si128(_mm_andnot_si128(tmp,  sc_mis_), _mm_and_si128(tmp,  sc_mch_));
+				tmp = _mm_or_si128(_mm_andnot_si128(mask, tmp),     _mm_and_si128(mask, sc_N_));
 #endif
-				tmp = _mm_andnot_si128(mask, tmp);
 				_mm_storeu_si128((__m128i*)((uint8_t*)s + t), tmp);
 			}
 		} else {
@@ -155,7 +169,7 @@ void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 #endif
 			}
 		} else if (!(flag&KSW_EZ_RIGHT)) { // gap left-alignment
-			__m128i *pr = p + r * n_col_ - st_;
+			__m128i *pr = p + (size_t)r * n_col_ - st_;
 			off[r] = st, off_end[r] = en;
 			for (t = st_; t <= en_; ++t) {
 				__m128i d, z, a, b, xt1, vt1, ut, tmp;
@@ -181,7 +195,7 @@ void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 				_mm_store_si128(&pr[t], d);
 			}
 		} else { // gap right-alignment
-			__m128i *pr = p + r * n_col_ - st_;
+			__m128i *pr = p + (size_t)r * n_col_ - st_;
 			off[r] = st, off_end[r] = en;
 			for (t = st_; t <= en_; ++t) {
 				__m128i d, z, a, b, xt1, vt1, ut, tmp;
@@ -277,10 +291,14 @@ void ksw_extz2_sse(void *km, int qlen, const uint8_t *query, int tlen, const uin
 	if (!approx_max) kfree(km, H);
 	if (with_cigar) { // backtrack
 		int rev_cigar = !!(flag & KSW_EZ_REV_CIGAR);
-		if (!ez->zdropped && !(flag&KSW_EZ_EXTZ_ONLY))
+		if (!ez->zdropped && !(flag&KSW_EZ_EXTZ_ONLY)) {
 			ksw_backtrack(km, 1, rev_cigar, 0, (uint8_t*)p, off, off_end, n_col_*16, tlen-1, qlen-1, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
-		else if (ez->max_t >= 0 && ez->max_q >= 0)
+		} else if (!ez->zdropped && (flag&KSW_EZ_EXTZ_ONLY) && ez->mqe + end_bonus > (int)ez->max) {
+			ez->reach_end = 1;
+			ksw_backtrack(km, 1, rev_cigar, 0, (uint8_t*)p, off, off_end, n_col_*16, ez->mqe_t, qlen-1, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
+		} else if (ez->max_t >= 0 && ez->max_q >= 0) {
 			ksw_backtrack(km, 1, rev_cigar, 0, (uint8_t*)p, off, off_end, n_col_*16, ez->max_t, ez->max_q, &ez->m_cigar, &ez->n_cigar, &ez->cigar);
+		}
 		kfree(km, mem2); kfree(km, off);
 	}
 }

ksw2_ll_sse.c

@@ -122,7 +122,7 @@ int ksw_ll_i16(void *q_, int tlen, const uint8_t *target, int _gapo, int _gape,
 			f = _mm_max_epi16(f, h);
 			h = _mm_load_si128(H0 + j);
 		}
-		for (k = 0; LIKELY(k < 16); ++k) {
+		for (k = 0; LIKELY(k < 8); ++k) {
 			f = _mm_slli_si128(f, 2);
 			for (j = 0; LIKELY(j < slen); ++j) {
 				h = _mm_load_si128(H1 + j);

kthread.c

@@ -1,6 +1,12 @@
 #include <pthread.h>
 #include <stdlib.h>
 #include <limits.h>
+#include <stdint.h>
+#include "kthread.h"
+
+#if (defined(WIN32) || defined(_WIN32)) && defined(_MSC_VER)
+#define __sync_fetch_and_add(ptr, addend)     _InterlockedExchangeAdd((void*)ptr, addend)
+#endif
 
 /************
  * kt_for() *
@@ -52,12 +58,13 @@ void kt_for(int n_threads, void (*func)(void*,long,int), void *data, long n)
 		kt_for_t t;
 		pthread_t *tid;
 		t.func = func, t.data = data, t.n_threads = n_threads, t.n = n;
-		t.w = (ktf_worker_t*)alloca(n_threads * sizeof(ktf_worker_t));
-		tid = (pthread_t*)alloca(n_threads * sizeof(pthread_t));
+		t.w = (ktf_worker_t*)calloc(n_threads, sizeof(ktf_worker_t));
+		tid = (pthread_t*)calloc(n_threads, sizeof(pthread_t));
 		for (i = 0; i < n_threads; ++i)
 			t.w[i].t = &t, t.w[i].i = i;
 		for (i = 0; i < n_threads; ++i) pthread_create(&tid[i], 0, ktf_worker, &t.w[i]);
 		for (i = 0; i < n_threads; ++i) pthread_join(tid[i], 0);
+		free(tid); free(t.w);
 	} else {
 		long j;
 		for (j = 0; j < n; ++j) func(data, j, 0);
@@ -135,16 +142,17 @@ void kt_pipeline(int n_threads, void *(*func)(void*, int, void*), void *shared_d
 	pthread_mutex_init(&aux.mutex, 0);
 	pthread_cond_init(&aux.cv, 0);
 
-	aux.workers = (ktp_worker_t*)alloca(n_threads * sizeof(ktp_worker_t));
+	aux.workers = (ktp_worker_t*)calloc(n_threads, sizeof(ktp_worker_t));
 	for (i = 0; i < n_threads; ++i) {
 		ktp_worker_t *w = &aux.workers[i];
 		w->step = 0; w->pl = &aux; w->data = 0;
 		w->index = aux.index++;
 	}
 
-	tid = (pthread_t*)alloca(n_threads * sizeof(pthread_t));
+	tid = (pthread_t*)calloc(n_threads, sizeof(pthread_t));
 	for (i = 0; i < n_threads; ++i) pthread_create(&tid[i], 0, ktp_worker, &aux.workers[i]);
 	for (i = 0; i < n_threads; ++i) pthread_join(tid[i], 0);
+	free(tid); free(aux.workers);
 
 	pthread_mutex_destroy(&aux.mutex);
 	pthread_cond_destroy(&aux.cv);

main.c

@@ -1,276 +1,379 @@
-#include <getopt.h>
 #include <stdlib.h>
 #include <stdio.h>
 #include <string.h>
-#include <sys/resource.h>
-#include <sys/time.h>
 #include "bseq.h"
 #include "minimap.h"
 #include "mmpriv.h"
+#include "ketopt.h"
 
-#define MM_VERSION "2.1-r311"
+#define MM_VERSION "2.13-r850"
 
+#ifdef __linux__
+#include <sys/resource.h>
+#include <sys/time.h>
 void liftrlimit()
 {
-#ifdef __linux__
 	struct rlimit r;
 	getrlimit(RLIMIT_AS, &r);
 	r.rlim_cur = r.rlim_max;
 	setrlimit(RLIMIT_AS, &r);
-#endif
 }
+#else
+void liftrlimit() {}
+#endif
 
-static struct option long_options[] = {
-	{ "bucket-bits",    required_argument, 0, 0 },
-	{ "mb-size",        required_argument, 0, 'K' },
-	{ "int-rname",      no_argument,       0, 0 },
-	{ "no-kalloc",      no_argument,       0, 0 },
-	{ "print-qname",    no_argument,       0, 0 },
-	{ "no-self",        no_argument,       0, 0 },
-	{ "print-seed",     no_argument,       0, 0 },
-	{ "max-chain-skip", required_argument, 0, 0 },
-	{ "min-dp-len",     required_argument, 0, 0 },
-	{ "print-aln-seq",  no_argument,       0, 0 },
-	{ "splice",         no_argument,       0, 0 },
-	{ "cost-non-gt-ag", required_argument, 0, 0 },
-	{ "no-sam-sq",      no_argument,       0, 0 },
-	{ "help",           no_argument,       0, 'h' },
-	{ "max-intron-len", required_argument, 0, 'G' },
-	{ "version",        no_argument,       0, 'V' },
-	{ "min-count",      required_argument, 0, 'n' },
-	{ "min-chain-score",required_argument, 0, 'm' },
-	{ "mask-level",     required_argument, 0, 'M' },
-	{ "min-dp-score",   required_argument, 0, 's' },
-	{ "sam",            no_argument,       0, 'a' },
-	{ 0, 0, 0, 0}
+static ko_longopt_t long_options[] = {
+	{ "bucket-bits",    ko_required_argument, 300 },
+	{ "mb-size",        ko_required_argument, 'K' },
+	{ "seed",           ko_required_argument, 302 },
+	{ "no-kalloc",      ko_no_argument,       303 },
+	{ "print-qname",    ko_no_argument,       304 },
+	{ "no-self",        ko_no_argument,       'D' },
+	{ "print-seeds",    ko_no_argument,       306 },
+	{ "max-chain-skip", ko_required_argument, 307 },
+	{ "min-dp-len",     ko_required_argument, 308 },
+	{ "print-aln-seq",  ko_no_argument,       309 },
+	{ "splice",         ko_no_argument,       310 },
+	{ "cost-non-gt-ag", ko_required_argument, 'C' },
+	{ "no-long-join",   ko_no_argument,       312 },
+	{ "sr",             ko_no_argument,       313 },
+	{ "frag",           ko_required_argument, 314 },
+	{ "secondary",      ko_required_argument, 315 },
+	{ "cs",             ko_optional_argument, 316 },
+	{ "end-bonus",      ko_required_argument, 317 },
+	{ "no-pairing",     ko_no_argument,       318 },
+	{ "splice-flank",   ko_required_argument, 319 },
+	{ "idx-no-seq",     ko_no_argument,       320 },
+	{ "end-seed-pen",   ko_required_argument, 321 },
+	{ "for-only",       ko_no_argument,       322 },
+	{ "rev-only",       ko_no_argument,       323 },
+	{ "heap-sort",      ko_required_argument, 324 },
+	{ "all-chain",      ko_no_argument,       'P' },
+	{ "dual",           ko_required_argument, 326 },
+	{ "max-clip-ratio", ko_required_argument, 327 },
+	{ "min-occ-floor",  ko_required_argument, 328 },
+	{ "MD",             ko_no_argument,       329 },
+	{ "lj-min-ratio",   ko_required_argument, 330 },
+	{ "score-N",        ko_required_argument, 331 },
+	{ "eqx",            ko_no_argument,       332 },
+	{ "paf-no-hit",     ko_no_argument,       333 },
+	{ "split-prefix",   ko_required_argument, 334 },
+	{ "no-end-flt",     ko_no_argument,       335 },
+	{ "hard-mask-level",ko_no_argument,       336 },
+	{ "help",           ko_no_argument,       'h' },
+	{ "max-intron-len", ko_required_argument, 'G' },
+	{ "version",        ko_no_argument,       'V' },
+	{ "min-count",      ko_required_argument, 'n' },
+	{ "min-chain-score",ko_required_argument, 'm' },
+	{ "mask-level",     ko_required_argument, 'M' },
+	{ "min-dp-score",   ko_required_argument, 's' },
+	{ "sam",            ko_no_argument,       'a' },
+	{ 0, 0, 0 }
 };
 
 static inline int64_t mm_parse_num(const char *str)
 {
 	double x;
 	char *p;
-	x = strtod(optarg, &p);
+	x = strtod(str, &p);
 	if (*p == 'G' || *p == 'g') x *= 1e9;
 	else if (*p == 'M' || *p == 'm') x *= 1e6;
 	else if (*p == 'K' || *p == 'k') x *= 1e3;
 	return (int64_t)(x + .499);
 }
 
+static inline void yes_or_no(mm_mapopt_t *opt, int flag, int long_idx, const char *arg, int yes_to_set)
+{
+	if (yes_to_set) {
+		if (strcmp(arg, "yes") == 0 || strcmp(arg, "y") == 0) opt->flag |= flag;
+		else if (strcmp(arg, "no") == 0 || strcmp(arg, "n") == 0) opt->flag &= ~flag;
+		else fprintf(stderr, "[WARNING]\033[1;31m option '--%s' only accepts 'yes' or 'no'.\033[0m\n", long_options[long_idx].name);
+	} else {
+		if (strcmp(arg, "yes") == 0 || strcmp(arg, "y") == 0) opt->flag &= ~flag;
+		else if (strcmp(arg, "no") == 0 || strcmp(arg, "n") == 0) opt->flag |= flag;
+		else fprintf(stderr, "[WARNING]\033[1;31m option '--%s' only accepts 'yes' or 'no'.\033[0m\n", long_options[long_idx].name);
+	}
+}
+
 int main(int argc, char *argv[])
 {
+	const char *opt_str = "2aSDw:k:K:t:r:f:Vv:g:G:I:d:XT:s:x:Hcp:M:n:z:A:B:O:E:m:N:Qu:R:hF:LC:yYP";
+	ketopt_t o = KETOPT_INIT;
 	mm_mapopt_t opt;
-	int i, c, k = 15, w = -1, bucket_bits = MM_IDX_DEF_B, n_threads = 3, keep_name = 1, is_idx, is_hpc = 0, long_idx, idx_par_set = 0, max_intron_len = 0, n_idx_part = 0;
-	int minibatch_size = 200000000;
-	uint64_t batch_size = 4000000000ULL;
-	mm_bseq_file_t *fp = 0;
+	mm_idxopt_t ipt;
+	int i, c, n_threads = 3, n_parts, old_best_n = -1;
 	char *fnw = 0, *rg = 0, *s;
-	FILE *fpr = 0, *fpw = 0, *fp_help = stderr;
+	FILE *fp_help = stderr;
+	mm_idx_reader_t *idx_rdr;
+	mm_idx_t *mi;
 
+	mm_verbose = 3;
 	liftrlimit();
 	mm_realtime0 = realtime();
-	mm_mapopt_init(&opt);
+	mm_set_opt(0, &ipt, &opt);
 
-	while ((c = getopt_long(argc, argv, "aSw:k:K:t:r:f:Vv:g:G:I:d:XT:s:x:Hcp:M:n:z:A:B:O:E:m:N:Qu:R:h", long_options, &long_idx)) >= 0) {
-		if (c == 'w') w = atoi(optarg), idx_par_set = 1;
-		else if (c == 'k') k = atoi(optarg), idx_par_set = 1;
-		else if (c == 'H') is_hpc = 1, idx_par_set = 1;
-		else if (c == 'd') fnw = optarg; // the above are indexing related options, except -I
-		else if (c == 'r') opt.bw = (int)mm_parse_num(optarg);
-		else if (c == 'f') opt.mid_occ_frac = atof(optarg);
-		else if (c == 't') n_threads = atoi(optarg);
-		else if (c == 'v') mm_verbose = atoi(optarg);
-		else if (c == 'g') opt.max_gap = (int)mm_parse_num(optarg);
-		else if (c == 'G') max_intron_len = (int)mm_parse_num(optarg);
-		else if (c == 'N') opt.best_n = atoi(optarg);
-		else if (c == 'p') opt.pri_ratio = atof(optarg);
-		else if (c == 'M') opt.mask_level = atof(optarg);
-		else if (c == 'c') opt.flag |= MM_F_OUT_CG | MM_F_CIGAR;
-		else if (c == 'S') opt.flag |= MM_F_OUT_CS | MM_F_CIGAR;
-		else if (c == 'X') opt.flag |= MM_F_AVA | MM_F_NO_SELF;
-		else if (c == 'a') opt.flag |= MM_F_OUT_SAM | MM_F_CIGAR;
-		else if (c == 'Q') opt.flag |= MM_F_NO_QUAL;
-		else if (c == 'T') opt.sdust_thres = atoi(optarg);
-		else if (c == 'n') opt.min_cnt = atoi(optarg);
-		else if (c == 'm') opt.min_chain_score = atoi(optarg);
-		else if (c == 'A') opt.a = atoi(optarg);
-		else if (c == 'B') opt.b = atoi(optarg);
-		else if (c == 'z') opt.zdrop = atoi(optarg);
-		else if (c == 's') opt.min_dp_max = atoi(optarg);
-		else if (c == 'I') batch_size = mm_parse_num(optarg);
-		else if (c == 'K') minibatch_size = (int)mm_parse_num(optarg);
-		else if (c == 'R') rg = optarg;
-		else if (c == 'h') fp_help = stdout;
-		else if (c == 0 && long_idx == 0) bucket_bits = atoi(optarg); // --bucket-bits
-		else if (c == 0 && long_idx == 2) keep_name = 0; // --int-rname
-		else if (c == 0 && long_idx == 3) mm_dbg_flag |= MM_DBG_NO_KALLOC; // --no-kalloc
-		else if (c == 0 && long_idx == 4) mm_dbg_flag |= MM_DBG_PRINT_QNAME; // --print-qname
-		else if (c == 0 && long_idx == 5) opt.flag |= MM_F_NO_SELF; // --no-self
-		else if (c == 0 && long_idx == 6) mm_dbg_flag |= MM_DBG_PRINT_QNAME | MM_DBG_PRINT_SEED; // --print-seed
-		else if (c == 0 && long_idx == 7) opt.max_chain_skip = atoi(optarg); // --max-chain-skip
-		else if (c == 0 && long_idx == 8) opt.min_ksw_len = atoi(optarg); // --min-dp-len
-		else if (c == 0 && long_idx == 9) mm_dbg_flag |= MM_DBG_PRINT_QNAME | MM_DBG_PRINT_ALN_SEQ; // --print-aln-seq
-		else if (c == 0 && long_idx ==10) opt.flag |= MM_F_SPLICE; // --splice
-		else if (c == 0 && long_idx ==11) opt.noncan = atoi(optarg); // --cost-non-gt-ag
-		else if (c == 0 && long_idx ==12) opt.flag |= MM_F_NO_SAM_SQ; // --no-sam-sq
-		else if (c == 'V') {
-			puts(MM_VERSION);
-			return 0;
-		} else if (c == 'u') {
-			if (*optarg == 'b') opt.flag |= MM_F_SPLICE_FOR|MM_F_SPLICE_REV;
-			else if (*optarg == 'B') opt.flag |= MM_F_SPLICE_BOTH;
-			else if (*optarg == 'f') opt.flag |= MM_F_SPLICE_FOR, opt.flag &= ~MM_F_SPLICE_REV;
-			else if (*optarg == 'r') opt.flag |= MM_F_SPLICE_REV, opt.flag &= ~MM_F_SPLICE_FOR;
-			else if (*optarg == 'n') opt.flag &= ~(MM_F_SPLICE_FOR|MM_F_SPLICE_REV);
-			else {
-				fprintf(stderr, "[E::%s] unrecognized cDNA direction\n", __func__);
-				return 1;
-			}
-		} else if (c == 'O') {
-			opt.q = opt.q2 = strtol(optarg, &s, 10);
-			if (*s == ',') opt.q2 = strtol(s + 1, &s, 10);
-		} else if (c == 'E') {
-			opt.e = opt.e2 = strtol(optarg, &s, 10);
-			if (*s == ',') opt.e2 = strtol(s + 1, &s, 10);
-		} else if (c == 'x') {
-			if (strcmp(optarg, "ava-ont") == 0) {
-				opt.flag |= MM_F_AVA | MM_F_NO_SELF;
-				opt.min_chain_score = 100, opt.pri_ratio = 0.0f, opt.max_gap = 10000, opt.max_chain_skip = 25;
-				minibatch_size = 500000000;
-				k = 15, w = 5;
-			} else if (strcmp(optarg, "ava-pb") == 0) {
-				opt.flag |= MM_F_AVA | MM_F_NO_SELF;
-				opt.min_chain_score = 100, opt.pri_ratio = 0.0f, opt.max_gap = 10000, opt.max_chain_skip = 25;
-				minibatch_size = 500000000;
-				is_hpc = 1, k = 19, w = 5;
-			} else if (strcmp(optarg, "map10k") == 0 || strcmp(optarg, "map-pb") == 0) {
-				is_hpc = 1, k = 19;
-			} else if (strcmp(optarg, "map-ont") == 0) {
-				is_hpc = 0, k = 15;
-			} else if (strcmp(optarg, "asm5") == 0) {
-				k = 19, w = 19;
-				opt.a = 1, opt.b = 19, opt.q = 39, opt.q2 = 81, opt.e = 3, opt.e2 = 1, opt.zdrop = 200;
-				opt.min_dp_max = 200;
-			} else if (strcmp(optarg, "asm10") == 0) {
-				k = 19, w = 19;
-				opt.a = 1, opt.b = 9, opt.q = 16, opt.q2 = 41, opt.e = 2, opt.e2 = 1, opt.zdrop = 200;
-				opt.min_dp_max = 200;
-			} else if (strcmp(optarg, "splice") == 0 || strcmp(optarg, "cdna") == 0) {
-				k = 15, w = 5;
-				opt.flag |= MM_F_SPLICE | MM_F_SPLICE_FOR | MM_F_SPLICE_REV;
-				opt.max_gap = 2000, opt.max_gap_ref = opt.bw = 200000;
-				opt.a = 1, opt.b = 2, opt.q = 2, opt.e = 1, opt.q2 = 32, opt.e2 = 0;
-				opt.noncan = 5;
-				opt.zdrop = 200;
-			} else {
-				fprintf(stderr, "[E::%s] unknown preset '%s'\n", __func__, optarg);
+	while ((c = ketopt(&o, argc, argv, 1, opt_str, long_options)) >= 0) { // test command line options and apply option -x/preset first
+		if (c == 'x') {
+			if (mm_set_opt(o.arg, &ipt, &opt) < 0) {
+				fprintf(stderr, "[ERROR] unknown preset '%s'\n", o.arg);
 				return 1;
 			}
+		} else if (c == ':') {
+			fprintf(stderr, "[ERROR] missing option argument\n");
+			return 1;
+		} else if (c == '?') {
+			fprintf(stderr, "[ERROR] unknown option in \"%s\"\n", argv[o.i]);
+			return 1;
 		}
 	}
-	if (w < 0) w = (int)(.6666667 * k + .499);
-	if ((opt.flag & MM_F_SPLICE) && max_intron_len > 0)
-		opt.max_gap_ref = opt.bw = max_intron_len;
+	o = KETOPT_INIT;
 
-	if (argc == optind || fp_help == stdout) {
+	while ((c = ketopt(&o, argc, argv, 1, opt_str, long_options)) >= 0) {
+		if (c == 'w') ipt.w = atoi(o.arg);
+		else if (c == 'k') ipt.k = atoi(o.arg);
+		else if (c == 'H') ipt.flag |= MM_I_HPC;
+		else if (c == 'd') fnw = o.arg; // the above are indexing related options, except -I
+		else if (c == 'r') opt.bw = (int)mm_parse_num(o.arg);
+		else if (c == 't') n_threads = atoi(o.arg);
+		else if (c == 'v') mm_verbose = atoi(o.arg);
+		else if (c == 'g') opt.max_gap = (int)mm_parse_num(o.arg);
+		else if (c == 'G') mm_mapopt_max_intron_len(&opt, (int)mm_parse_num(o.arg));
+		else if (c == 'F') opt.max_frag_len = (int)mm_parse_num(o.arg);
+		else if (c == 'N') old_best_n = opt.best_n, opt.best_n = atoi(o.arg);
+		else if (c == 'p') opt.pri_ratio = atof(o.arg);
+		else if (c == 'M') opt.mask_level = atof(o.arg);
+		else if (c == 'c') opt.flag |= MM_F_OUT_CG | MM_F_CIGAR;
+		else if (c == 'D') opt.flag |= MM_F_NO_DIAG;
+		else if (c == 'P') opt.flag |= MM_F_ALL_CHAINS;
+		else if (c == 'X') opt.flag |= MM_F_ALL_CHAINS | MM_F_NO_DIAG | MM_F_NO_DUAL | MM_F_NO_LJOIN; // -D -P --no-long-join --dual=no
+		else if (c == 'a') opt.flag |= MM_F_OUT_SAM | MM_F_CIGAR;
+		else if (c == 'Q') opt.flag |= MM_F_NO_QUAL;
+		else if (c == 'Y') opt.flag |= MM_F_SOFTCLIP;
+		else if (c == 'L') opt.flag |= MM_F_LONG_CIGAR;
+		else if (c == 'y') opt.flag |= MM_F_COPY_COMMENT;
+		else if (c == 'T') opt.sdust_thres = atoi(o.arg);
+		else if (c == 'n') opt.min_cnt = atoi(o.arg);
+		else if (c == 'm') opt.min_chain_score = atoi(o.arg);
+		else if (c == 'A') opt.a = atoi(o.arg);
+		else if (c == 'B') opt.b = atoi(o.arg);
+		else if (c == 's') opt.min_dp_max = atoi(o.arg);
+		else if (c == 'C') opt.noncan = atoi(o.arg);
+		else if (c == 'I') ipt.batch_size = mm_parse_num(o.arg);
+		else if (c == 'K') opt.mini_batch_size = (int)mm_parse_num(o.arg);
+		else if (c == 'R') rg = o.arg;
+		else if (c == 'h') fp_help = stdout;
+		else if (c == '2') opt.flag |= MM_F_2_IO_THREADS;
+		else if (c == 300) ipt.bucket_bits = atoi(o.arg); // --bucket-bits
+		else if (c == 302) opt.seed = atoi(o.arg); // --seed
+		else if (c == 303) mm_dbg_flag |= MM_DBG_NO_KALLOC; // --no-kalloc
+		else if (c == 304) mm_dbg_flag |= MM_DBG_PRINT_QNAME; // --print-qname
+		else if (c == 306) mm_dbg_flag |= MM_DBG_PRINT_QNAME | MM_DBG_PRINT_SEED, n_threads = 1; // --print-seed
+		else if (c == 307) opt.max_chain_skip = atoi(o.arg); // --max-chain-skip
+		else if (c == 308) opt.min_ksw_len = atoi(o.arg); // --min-dp-len
+		else if (c == 309) mm_dbg_flag |= MM_DBG_PRINT_QNAME | MM_DBG_PRINT_ALN_SEQ, n_threads = 1; // --print-aln-seq
+		else if (c == 310) opt.flag |= MM_F_SPLICE; // --splice
+		else if (c == 312) opt.flag |= MM_F_NO_LJOIN; // --no-long-join
+		else if (c == 313) opt.flag |= MM_F_SR; // --sr
+		else if (c == 317) opt.end_bonus = atoi(o.arg); // --end-bonus
+		else if (c == 318) opt.flag |= MM_F_INDEPEND_SEG; // --no-pairing
+		else if (c == 320) ipt.flag |= MM_I_NO_SEQ; // --idx-no-seq
+		else if (c == 321) opt.anchor_ext_shift = atoi(o.arg); // --end-seed-pen
+		else if (c == 322) opt.flag |= MM_F_FOR_ONLY; // --for-only
+		else if (c == 323) opt.flag |= MM_F_REV_ONLY; // --rev-only
+		else if (c == 327) opt.max_clip_ratio = atof(o.arg); // --max-clip-ratio
+		else if (c == 328) opt.min_mid_occ = atoi(o.arg); // --min-occ-floor
+		else if (c == 329) opt.flag |= MM_F_OUT_MD; // --MD
+		else if (c == 330) opt.min_join_flank_ratio = atof(o.arg); // --lj-min-ratio
+		else if (c == 331) opt.sc_ambi = atoi(o.arg); // --score-N
+		else if (c == 332) opt.flag |= MM_F_EQX; // --eqx
+		else if (c == 333) opt.flag |= MM_F_PAF_NO_HIT; // --paf-no-hit
+		else if (c == 334) opt.split_prefix = o.arg; // --split-prefix
+		else if (c == 335) opt.flag |= MM_F_NO_END_FLT; // --no-end-flt
+		else if (c == 336) opt.flag |= MM_F_HARD_MLEVEL; // --hard-mask-level
+		else if (c == 314) { // --frag
+			yes_or_no(&opt, MM_F_FRAG_MODE, o.longidx, o.arg, 1);
+		} else if (c == 315) { // --secondary
+			yes_or_no(&opt, MM_F_NO_PRINT_2ND, o.longidx, o.arg, 0);
+		} else if (c == 316) { // --cs
+			opt.flag |= MM_F_OUT_CS | MM_F_CIGAR;
+			if (o.arg == 0 || strcmp(o.arg, "short") == 0) {
+				opt.flag &= ~MM_F_OUT_CS_LONG;
+			} else if (strcmp(o.arg, "long") == 0) {
+				opt.flag |= MM_F_OUT_CS_LONG;
+			} else if (strcmp(o.arg, "none") == 0) {
+				opt.flag &= ~MM_F_OUT_CS;
+			} else if (mm_verbose >= 2) {
+				fprintf(stderr, "[WARNING]\033[1;31m --cs only takes 'short' or 'long'. Invalid values are assumed to be 'short'.\033[0m\n");
+			}
+		} else if (c == 319) { // --splice-flank
+			yes_or_no(&opt, MM_F_SPLICE_FLANK, o.longidx, o.arg, 1);
+		} else if (c == 324) { // --heap-sort
+			yes_or_no(&opt, MM_F_HEAP_SORT, o.longidx, o.arg, 1);
+		} else if (c == 326) { // --dual
+			yes_or_no(&opt, MM_F_NO_DUAL, o.longidx, o.arg, 0);
+		} else if (c == 'S') {
+			opt.flag |= MM_F_OUT_CS | MM_F_CIGAR | MM_F_OUT_CS_LONG;
+			if (mm_verbose >= 2)
+				fprintf(stderr, "[WARNING]\033[1;31m option -S is deprecated and may be removed in future. Please use --cs=long instead.\033[0m\n");
+		} else if (c == 'V') {
+			puts(MM_VERSION);
+			return 0;
+		} else if (c == 'f') {
+			double x;
+			char *p;
+			x = strtod(o.arg, &p);
+			if (x < 1.0) opt.mid_occ_frac = x, opt.mid_occ = 0;
+			else opt.mid_occ = (int)(x + .499);
+			if (*p == ',') opt.max_occ = (int)(strtod(p+1, &p) + .499);
+		} else if (c == 'u') {
+			if (*o.arg == 'b') opt.flag |= MM_F_SPLICE_FOR|MM_F_SPLICE_REV; // both strands
+			else if (*o.arg == 'f') opt.flag |= MM_F_SPLICE_FOR, opt.flag &= ~MM_F_SPLICE_REV; // match GT-AG
+			else if (*o.arg == 'r') opt.flag |= MM_F_SPLICE_REV, opt.flag &= ~MM_F_SPLICE_FOR; // match CT-AC (reverse complement of GT-AG)
+			else if (*o.arg == 'n') opt.flag &= ~(MM_F_SPLICE_FOR|MM_F_SPLICE_REV); // don't try to match the GT-AG signal
+			else {
+				fprintf(stderr, "[ERROR]\033[1;31m unrecognized cDNA direction\033[0m\n");
+				return 1;
+			}
+		} else if (c == 'z') {
+			opt.zdrop = opt.zdrop_inv = strtol(o.arg, &s, 10);
+			if (*s == ',') opt.zdrop_inv = strtol(s + 1, &s, 10);
+		} else if (c == 'O') {
+			opt.q = opt.q2 = strtol(o.arg, &s, 10);
+			if (*s == ',') opt.q2 = strtol(s + 1, &s, 10);
+		} else if (c == 'E') {
+			opt.e = opt.e2 = strtol(o.arg, &s, 10);
+			if (*s == ',') opt.e2 = strtol(s + 1, &s, 10);
+		}
+	}
+	if ((opt.flag & MM_F_SPLICE) && (opt.flag & MM_F_FRAG_MODE)) {
+		fprintf(stderr, "[ERROR]\033[1;31m --splice and --frag should not be specified at the same time.\033[0m\n");
+		return 1;
+	}
+	if (!fnw && !(opt.flag&MM_F_CIGAR))
+		ipt.flag |= MM_I_NO_SEQ;
+	if (mm_check_opt(&ipt, &opt) < 0)
+		return 1;
+	if (opt.best_n == 0) {
+		fprintf(stderr, "[WARNING]\033[1;31m changed '-N 0' to '-N %d --secondary=no'.\033[0m\n", old_best_n);
+		opt.best_n = old_best_n, opt.flag |= MM_F_NO_PRINT_2ND;
+	}
+
+	if (argc == o.ind || fp_help == stdout) {
 		fprintf(fp_help, "Usage: minimap2 [options] <target.fa>|<target.idx> [query.fa] [...]\n");
 		fprintf(fp_help, "Options:\n");
 		fprintf(fp_help, "  Indexing:\n");
-		fprintf(fp_help, "    -H           use homopolymer-compressed k-mer\n");
-		fprintf(fp_help, "    -k INT       k-mer size (no larger than 28) [%d]\n", k);
-		fprintf(fp_help, "    -w INT       minizer window size [{-k}*2/3]\n");
+		fprintf(fp_help, "    -H           use homopolymer-compressed k-mer (preferrable for PacBio)\n");
+		fprintf(fp_help, "    -k INT       k-mer size (no larger than 28) [%d]\n", ipt.k);
+		fprintf(fp_help, "    -w INT       minizer window size [%d]\n", ipt.w);
 		fprintf(fp_help, "    -I NUM       split index for every ~NUM input bases [4G]\n");
 		fprintf(fp_help, "    -d FILE      dump index to FILE []\n");
 		fprintf(fp_help, "  Mapping:\n");
 		fprintf(fp_help, "    -f FLOAT     filter out top FLOAT fraction of repetitive minimizers [%g]\n", opt.mid_occ_frac);
-		fprintf(fp_help, "    -g INT       stop chain enlongation if there are no minimizers in INT-bp [%d]\n", opt.max_gap);
-		fprintf(fp_help, "    -r INT       bandwidth used in chaining and DP-based alignment [%d]\n", opt.bw);
+		fprintf(fp_help, "    -g NUM       stop chain enlongation if there are no minimizers in INT-bp [%d]\n", opt.max_gap);
+		fprintf(fp_help, "    -G NUM       max intron length (effective with -xsplice; changing -r) [200k]\n");
+		fprintf(fp_help, "    -F NUM       max fragment length (effective with -xsr or in the fragment mode) [800]\n");
+		fprintf(fp_help, "    -r NUM       bandwidth used in chaining and DP-based alignment [%d]\n", opt.bw);
 		fprintf(fp_help, "    -n INT       minimal number of minimizers on a chain [%d]\n", opt.min_cnt);
 		fprintf(fp_help, "    -m INT       minimal chaining score (matching bases minus log gap penalty) [%d]\n", opt.min_chain_score);
 //		fprintf(fp_help, "    -T INT       SDUST threshold; 0 to disable SDUST [%d]\n", opt.sdust_thres); // TODO: this option is never used; might be buggy
 		fprintf(fp_help, "    -X           skip self and dual mappings (for the all-vs-all mode)\n");
 		fprintf(fp_help, "    -p FLOAT     min secondary-to-primary score ratio [%g]\n", opt.pri_ratio);
 		fprintf(fp_help, "    -N INT       retain at most INT secondary alignments [%d]\n", opt.best_n);
-		fprintf(fp_help, "    -G NUM       max intron length (only effective following -x splice) [200k]\n");
 		fprintf(fp_help, "  Alignment:\n");
 		fprintf(fp_help, "    -A INT       matching score [%d]\n", opt.a);
 		fprintf(fp_help, "    -B INT       mismatch penalty [%d]\n", opt.b);
 		fprintf(fp_help, "    -O INT[,INT] gap open penalty [%d,%d]\n", opt.q, opt.q2);
 		fprintf(fp_help, "    -E INT[,INT] gap extension penalty; a k-long gap costs min{O1+k*E1,O2+k*E2} [%d,%d]\n", opt.e, opt.e2);
-		fprintf(fp_help, "    -z INT       Z-drop score [%d]\n", opt.zdrop);
+		fprintf(fp_help, "    -z INT[,INT] Z-drop score and inversion Z-drop score [%d,%d]\n", opt.zdrop, opt.zdrop_inv);
 		fprintf(fp_help, "    -s INT       minimal peak DP alignment score [%d]\n", opt.min_dp_max);
 		fprintf(fp_help, "    -u CHAR      how to find GT-AG. f:transcript strand, b:both strands, n:don't match GT-AG [n]\n");
 		fprintf(fp_help, "  Input/Output:\n");
 		fprintf(fp_help, "    -a           output in the SAM format (PAF by default)\n");
 		fprintf(fp_help, "    -Q           don't output base quality in SAM\n");
+		fprintf(fp_help, "    -L           write CIGAR with >65535 ops at the CG tag\n");
 		fprintf(fp_help, "    -R STR       SAM read group line in a format like '@RG\\tID:foo\\tSM:bar' []\n");
 		fprintf(fp_help, "    -c           output CIGAR in PAF\n");
-		fprintf(fp_help, "    -S           output the cs tag in PAF (cs encodes both query and ref sequences)\n");
+		fprintf(fp_help, "    --cs[=STR]   output the cs tag; STR is 'short' (if absent) or 'long' [none]\n");
+		fprintf(fp_help, "    --MD         output the MD tag\n");
+		fprintf(fp_help, "    --eqx        write =/X CIGAR operators\n");
+		fprintf(fp_help, "    -Y           use soft clipping for supplementary alignments\n");
 		fprintf(fp_help, "    -t INT       number of threads [%d]\n", n_threads);
-		fprintf(fp_help, "    -K NUM       minibatch size [200M]\n");
+		fprintf(fp_help, "    -K NUM       minibatch size for mapping [500M]\n");
 //		fprintf(fp_help, "    -v INT       verbose level [%d]\n", mm_verbose);
 		fprintf(fp_help, "    --version    show version number\n");
 		fprintf(fp_help, "  Preset:\n");
-		fprintf(fp_help, "    -x STR       preset (recommended to be applied before other options) []\n");
-		fprintf(fp_help, "                 map10k/map-pb: -Hk19 (PacBio/ONT vs reference mapping)\n");
-		fprintf(fp_help, "                 map-ont: -k15 (slightly more sensitive than 'map10k' for ONT vs reference)\n");
-		fprintf(fp_help, "                 asm5: -k19 -w19 -A1 -B19 -O39,81 -E3,1 -s200 -z200 (asm to ref mapping; break at 5%% div.)\n");
-		fprintf(fp_help, "                 asm10: -k19 -w19 -A1 -B9 -O16,41 -E2,1 -s200 -z200 (asm to ref mapping; break at 10%% div.)\n");
-		fprintf(fp_help, "                 ava-pb: -Hk19 -w5 -Xp0 -m100 -g10000 -K500m --max-chain-skip 25 (PacBio read overlap)\n");
-		fprintf(fp_help, "                 ava-ont: -k15 -w5 -Xp0 -m100 -g10000 -K500m --max-chain-skip 25 (ONT read overlap)\n");
-		fprintf(fp_help, "                 splice: long-read spliced alignment (see minimap2.1 for details)\n");
-		fprintf(fp_help, "\nSee `man ./minimap2.1' for detailed description of command-line options.\n");
+		fprintf(fp_help, "    -x STR       preset (always applied before other options; see minimap2.1 for details) []\n");
+		fprintf(fp_help, "                 - map-pb/map-ont: PacBio/Nanopore vs reference mapping\n");
+		fprintf(fp_help, "                 - ava-pb/ava-ont: PacBio/Nanopore read overlap\n");
+		fprintf(fp_help, "                 - asm5/asm10/asm20: asm-to-ref mapping, for ~0.1/1/5%% sequence divergence\n");
+		fprintf(fp_help, "                 - splice: long-read spliced alignment\n");
+		fprintf(fp_help, "                 - sr: genomic short-read mapping\n");
+		fprintf(fp_help, "\nSee `man ./minimap2.1' for detailed description of these and other advanced command-line options.\n");
 		return fp_help == stdout? 0 : 1;
 	}
 
-	is_idx = mm_idx_is_idx(argv[optind]);
-	if (is_idx < 0) {
-		fprintf(stderr, "[ERROR] failed to open file '%s'\n", argv[optind]);
+	if ((opt.flag & MM_F_SR) && argc - o.ind > 3) {
+		fprintf(stderr, "[ERROR] incorrect input: in the sr mode, please specify no more than two query files.\n");
 		return 1;
 	}
-	if (!is_idx && fnw == 0 && argc - optind < 2) {
+	idx_rdr = mm_idx_reader_open(argv[o.ind], &ipt, fnw);
+	if (idx_rdr == 0) {
+		fprintf(stderr, "[ERROR] failed to open file '%s'\n", argv[o.ind]);
+		return 1;
+	}
+	if (!idx_rdr->is_idx && fnw == 0 && argc - o.ind < 2) {
 		fprintf(stderr, "[ERROR] missing input: please specify a query file to map or option -d to keep the index\n");
+		mm_idx_reader_close(idx_rdr);
 		return 1;
 	}
-	if (is_idx) fpr = fopen(argv[optind], "rb");
-	else fp = mm_bseq_open(argv[optind]);
-	if (fnw) fpw = fopen(fnw, "wb");
-	if (opt.flag & MM_F_OUT_SAM)
-		mm_write_sam_hdr_no_SQ(rg, MM_VERSION, argc, argv);
-	for (;;) {
-		mm_idx_t *mi;
-		if (fpr) {
-			mi = mm_idx_load(fpr);
-			if (idx_par_set && mm_verbose >= 2 && (mi->k != k || mi->w != w || mi->is_hpc != is_hpc))
-				fprintf(stderr, "[WARNING] \033[1;31mIndexing parameters on the command line (-k/-w/-H) overridden by parameters in the prebuilt index.\033[0m\n");
-		} else {
-			mi = mm_idx_gen(fp, w, k, bucket_bits, is_hpc, minibatch_size, n_threads, batch_size, keep_name);
+	if (opt.best_n == 0 && (opt.flag&MM_F_CIGAR) && mm_verbose >= 2)
+		fprintf(stderr, "[WARNING]\033[1;31m `-N 0' reduces alignment accuracy. Please use --secondary=no to suppress secondary alignments.\033[0m\n");
+	while ((mi = mm_idx_reader_read(idx_rdr, n_threads)) != 0) {
+		if ((opt.flag & MM_F_CIGAR) && (mi->flag & MM_I_NO_SEQ)) {
+			fprintf(stderr, "[ERROR] the prebuilt index doesn't contain sequences.\n");
+			mm_idx_destroy(mi);
+			mm_idx_reader_close(idx_rdr);
+			return 1;
+		}
+		if ((opt.flag & MM_F_OUT_SAM) && idx_rdr->n_parts == 1) {
+			if (mm_idx_reader_eof(idx_rdr)) {
+				mm_write_sam_hdr(mi, rg, MM_VERSION, argc, argv);
+			} else {
+				mm_write_sam_hdr(0, rg, MM_VERSION, argc, argv);
+				if (opt.split_prefix == 0 && mm_verbose >= 2)
+					fprintf(stderr, "[WARNING]\033[1;31m For a multi-part index, no @SQ lines will be outputted. Please use --split-prefix.\033[0m\n");
+			}
 		}
-		if (mi == 0) break;
-		++n_idx_part;
-		if (mm_verbose >= 2 && n_idx_part > 1 && (opt.flag&MM_F_OUT_SAM) && !(opt.flag&MM_F_NO_SAM_SQ))
-			fprintf(stderr, "[WARNING] \033[1;31mSAM output is malformated due to internal @SQ lines. Please add option --no-sam-sq or filter afterwards.\033[0m\n");
 		if (mm_verbose >= 3)
 			fprintf(stderr, "[M::%s::%.3f*%.2f] loaded/built the index for %d target sequence(s)\n",
 					__func__, realtime() - mm_realtime0, cputime() / (realtime() - mm_realtime0), mi->n_seq);
-		if (fpw) {
-			mm_idx_dump(fpw, mi);
-			if (mm_verbose >= 3)
-				fprintf(stderr, "[M::%s::%.3f*%.2f] dumpped the (partial) index to disk\n", __func__, realtime() - mm_realtime0, cputime() / (realtime() - mm_realtime0));
-		}
-		if (argc != optind + 1) mm_mapopt_update(&opt, mi);
+		if (argc != o.ind + 1) mm_mapopt_update(&opt, mi);
 		if (mm_verbose >= 3) mm_idx_stat(mi);
-		for (i = optind + 1; i < argc; ++i)
-			mm_map_file(mi, argv[i], &opt, n_threads, minibatch_size);
+		if (!(opt.flag & MM_F_FRAG_MODE)) {
+			for (i = o.ind + 1; i < argc; ++i)
+				mm_map_file(mi, argv[i], &opt, n_threads);
+		} else {
+			mm_map_file_frag(mi, argc - (o.ind + 1), (const char**)&argv[o.ind + 1], &opt, n_threads);
+		}
 		mm_idx_destroy(mi);
 	}
-	if (fpw) fclose(fpw);
-	if (fpr) fclose(fpr);
-	if (fp)  mm_bseq_close(fp);
+	n_parts = idx_rdr->n_parts;
+	mm_idx_reader_close(idx_rdr);
 
-	fprintf(stderr, "[M::%s] Version: %s\n", __func__, MM_VERSION);
-	fprintf(stderr, "[M::%s] CMD:", __func__);
-	for (i = 0; i < argc; ++i)
-		fprintf(stderr, " %s", argv[i]);
-	fprintf(stderr, "\n[M::%s] Real time: %.3f sec; CPU: %.3f sec\n", __func__, realtime() - mm_realtime0, cputime());
+	if (opt.split_prefix)
+		mm_split_merge(argc - (o.ind + 1), (const char**)&argv[o.ind + 1], &opt, n_parts);
+
+	if (fflush(stdout) == EOF) {
+		fprintf(stderr, "[ERROR] failed to write the results\n");
+		exit(EXIT_FAILURE);
+	}
+
+	if (mm_verbose >= 3) {
+		fprintf(stderr, "[M::%s] Version: %s\n", __func__, MM_VERSION);
+		fprintf(stderr, "[M::%s] CMD:", __func__);
+		for (i = 0; i < argc; ++i)
+			fprintf(stderr, " %s", argv[i]);
+		fprintf(stderr, "\n[M::%s] Real time: %.3f sec; CPU: %.3f sec; Peak RSS: %.3f GB\n", __func__, realtime() - mm_realtime0, cputime(), peakrss() / 1024.0 / 1024.0 / 1024.0);
+	}
 	return 0;
 }

map.c

@@ -7,59 +7,11 @@
 #include "sdust.h"
 #include "mmpriv.h"
 #include "bseq.h"
-
-void mm_mapopt_init(mm_mapopt_t *opt)
-{
-	memset(opt, 0, sizeof(mm_mapopt_t));
-	opt->max_occ_frac = 1e-5f;
-	opt->mid_occ_frac = 2e-4f;
-	opt->sdust_thres = 0;
-
-	opt->min_cnt = 3;
-	opt->min_chain_score = 40;
-	opt->bw = 500;
-	opt->max_gap = 5000;
-	opt->max_gap_ref = -1;
-	opt->max_chain_skip = 25;
-
-	opt->mask_level = 0.5f;
-	opt->pri_ratio = 0.8f;
-	opt->best_n = 5;
-
-	opt->max_join_long = 20000;
-	opt->max_join_short = 2000;
-	opt->min_join_flank_sc = 1000;
-
-	opt->a = 2, opt->b = 4, opt->q = 4, opt->e = 2, opt->q2 = 24, opt->e2 = 1;
-	opt->zdrop = 400;
-	opt->min_dp_max = opt->min_chain_score * opt->a;
-	opt->min_ksw_len = 200;
-}
-
-void mm_mapopt_update(mm_mapopt_t *opt, const mm_idx_t *mi)
-{
-	if (opt->flag & MM_F_SPLICE_BOTH)
-		opt->flag &= ~(MM_F_SPLICE_FOR|MM_F_SPLICE_REV);
-	opt->max_occ = mm_idx_cal_max_occ(mi, opt->max_occ_frac);
-	opt->mid_occ = mm_idx_cal_max_occ(mi, opt->mid_occ_frac);
-	if (mm_verbose >= 3)
-		fprintf(stderr, "[M::%s::%.3f*%.2f] mid_occ = %d; max_occ = %d\n", __func__, realtime() - mm_realtime0, cputime() / (realtime() - mm_realtime0),
-				opt->mid_occ, opt->max_occ);
-}
-
-typedef struct {
-	uint32_t n:31, is_alloc:1;
-	uint32_t qpos;
-	union {
-		const uint64_t *cr;
-		uint64_t *r;
-	} x;
-} mm_match_t;
+#include "khash.h"
 
 struct mm_tbuf_s {
-	sdust_buf_t *sdb;
-	mm128_v mini;
 	void *km;
+	int rep_len, frag_gap;
 };
 
 mm_tbuf_t *mm_tbuf_init(void)
@@ -67,226 +19,376 @@ mm_tbuf_t *mm_tbuf_init(void)
 	mm_tbuf_t *b;
 	b = (mm_tbuf_t*)calloc(1, sizeof(mm_tbuf_t));
 	if (!(mm_dbg_flag & 1)) b->km = km_init();
-	b->sdb = sdust_buf_init(b->km);
 	return b;
 }
 
 void mm_tbuf_destroy(mm_tbuf_t *b)
 {
 	if (b == 0) return;
-	kfree(b->km, b->mini.a);
-	sdust_buf_destroy(b->sdb);
 	km_destroy(b->km);
 	free(b);
 }
 
-static void mm_dust_minier(mm128_v *mini, int l_seq, const char *seq, int sdust_thres, sdust_buf_t *sdb)
+void *mm_tbuf_get_km(mm_tbuf_t *b)
+{
+	return b->km;
+}
+
+static int mm_dust_minier(void *km, int n, mm128_t *a, int l_seq, const char *seq, int sdust_thres)
 {
 	int n_dreg, j, k, u = 0;
 	const uint64_t *dreg;
-	if (sdust_thres <= 0 || sdb == 0) return;
+	sdust_buf_t *sdb;
+	if (sdust_thres <= 0) return n;
+	sdb = sdust_buf_init(km);
 	dreg = sdust_core((const uint8_t*)seq, l_seq, sdust_thres, 64, &n_dreg, sdb);
-	for (j = k = 0; j < mini->n; ++j) { // squeeze out minimizers that significantly overlap with LCRs
-		int32_t qpos = (uint32_t)mini->a[j].y>>1, span = mini->a[j].x&0xff;
+	for (j = k = 0; j < n; ++j) { // squeeze out minimizers that significantly overlap with LCRs
+		int32_t qpos = (uint32_t)a[j].y>>1, span = a[j].x&0xff;
 		int32_t s = qpos - (span - 1), e = s + span;
-		while (u < n_dreg && (uint32_t)dreg[u] <= s) ++u;
-		if (u < n_dreg && dreg[u]>>32 < e) {
+		while (u < n_dreg && (int32_t)dreg[u] <= s) ++u;
+		if (u < n_dreg && (int32_t)(dreg[u]>>32) < e) {
 			int v, l = 0;
-			for (v = u; v < n_dreg && dreg[v]>>32 < e; ++v) { // iterate over LCRs overlapping this minimizer
-				int ss = s > dreg[v]>>32? s : dreg[v]>>32;
-				int ee = e < (uint32_t)dreg[v]? e : (uint32_t)dreg[v];
+			for (v = u; v < n_dreg && (int32_t)(dreg[v]>>32) < e; ++v) { // iterate over LCRs overlapping this minimizer
+				int ss = s > (int32_t)(dreg[v]>>32)? s : dreg[v]>>32;
+				int ee = e < (int32_t)dreg[v]? e : (uint32_t)dreg[v];
 				l += ee - ss;
 			}
-			if (l <= span>>1) mini->a[k++] = mini->a[j]; // keep the minimizer if less than half of it falls in masked region
+			if (l <= span>>1) a[k++] = a[j]; // keep the minimizer if less than half of it falls in masked region
+		} else a[k++] = a[j];
+	}
+	sdust_buf_destroy(sdb);
+	return k; // the new size
+}
+
+static void collect_minimizers(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int n_segs, const int *qlens, const char **seqs, mm128_v *mv)
+{
+	int i, n, sum = 0;
+	mv->n = 0;
+	for (i = n = 0; i < n_segs; ++i) {
+		size_t j;
+		mm_sketch(km, seqs[i], qlens[i], mi->w, mi->k, i, mi->flag&MM_I_HPC, mv);
+		for (j = n; j < mv->n; ++j)
+			mv->a[j].y += sum << 1;
+		if (opt->sdust_thres > 0) // mask low-complexity minimizers
+			mv->n = n + mm_dust_minier(km, mv->n - n, mv->a + n, qlens[i], seqs[i], opt->sdust_thres);
+		sum += qlens[i], n = mv->n;
+	}
+}
+
+#include "ksort.h"
+#define heap_lt(a, b) ((a).x > (b).x)
+KSORT_INIT(heap, mm128_t, heap_lt)
+
+typedef struct {
+	uint32_t n;
+	uint32_t q_pos, q_span;
+	uint32_t seg_id:31, is_tandem:1;
+	const uint64_t *cr;
+} mm_match_t;
+
+static mm_match_t *collect_matches(void *km, int *_n_m, int max_occ, const mm_idx_t *mi, const mm128_v *mv, int64_t *n_a, int *rep_len, int *n_mini_pos, uint64_t **mini_pos)
+{
+	int rep_st = 0, rep_en = 0, n_m;
+	size_t i;
+	mm_match_t *m;
+	*n_mini_pos = 0;
+	*mini_pos = (uint64_t*)kmalloc(km, mv->n * sizeof(uint64_t));
+	m = (mm_match_t*)kmalloc(km, mv->n * sizeof(mm_match_t));
+	for (i = 0, n_m = 0, *rep_len = 0, *n_a = 0; i < mv->n; ++i) {
+		const uint64_t *cr;
+		mm128_t *p = &mv->a[i];
+		uint32_t q_pos = (uint32_t)p->y, q_span = p->x & 0xff;
+		int t;
+		cr = mm_idx_get(mi, p->x>>8, &t);
+		if (t >= max_occ) {
+			int en = (q_pos >> 1) + 1, st = en - q_span;
+			if (st > rep_en) {
+				*rep_len += rep_en - rep_st;
+				rep_st = st, rep_en = en;
+			} else rep_en = en;
+		} else {
+			mm_match_t *q = &m[n_m++];
+			q->q_pos = q_pos, q->q_span = q_span, q->cr = cr, q->n = t, q->seg_id = p->y >> 32;
+			q->is_tandem = 0;
+			if (i > 0 && p->x>>8 == mv->a[i - 1].x>>8) q->is_tandem = 1;
+			if (i < mv->n - 1 && p->x>>8 == mv->a[i + 1].x>>8) q->is_tandem = 1;
+			*n_a += q->n;
+			(*mini_pos)[(*n_mini_pos)++] = (uint64_t)q_span<<32 | q_pos>>1;
 		}
 	}
-	mini->n = k;
+	*rep_len += rep_en - rep_st;
+	*_n_m = n_m;
+	return m;
 }
-#if 0
-int mm_pair_thin_core(mm_tbuf_t *b, uint64_t x, int radius, int rel, int st0, int n, const uint64_t *z, uint64_v *a)
+
+static inline int skip_seed(int flag, uint64_t r, const mm_match_t *q, const char *qname, int qlen, const mm_idx_t *mi, int *is_self)
 {
-	int i, st = st0, en = n, mid = en - 1;
-	while (st < en) {
-		uint64_t y;
-		mid = st + ((en - st) >> 1);
-		y = z[mid];
-		if (y < x && (x - y)>>1 > radius) st = mid + 1;
-		else if (y >= x && (y - x)>>1 > radius) en = mid;
-		else break;
+	*is_self = 0;
+	if (qname && (flag & (MM_F_NO_DIAG|MM_F_NO_DUAL))) {
+		const mm_idx_seq_t *s = &mi->seq[r>>32];
+		int cmp;
+		cmp = strcmp(qname, s->name);
+		if ((flag&MM_F_NO_DIAG) && cmp == 0 && (int)s->len == qlen) {
+			if ((uint32_t)r>>1 == (q->q_pos>>1)) return 1; // avoid the diagnonal anchors
+			if ((r&1) == (q->q_pos&1)) *is_self = 1; // this flag is used to avoid spurious extension on self chain
+		}
+		if ((flag&MM_F_NO_DUAL) && cmp > 0) // all-vs-all mode: map once
+			return 1;
 	}
-	if (st < en) {
-		for (en = mid + 1; en < n; ++en)
-			if (z[en] > x && (z[en] - x)>>1 > radius)
-				break;
-		for (st = mid - 1; st >= st0; --st)
-			if (z[st] < x && (x - z[st])>>1 > radius)
-				break;
-		++st;
-		for (i = st; i < en; ++i) {
-			uint64_t y = z[i];
-			if (((x ^ y) & 1) == rel) {
-//				printf("* %d,%d\n", (uint32_t)x>>1, (uint32_t)y>>1);
-				kv_push(uint64_t, b->km, *a, y);
+	if (flag & (MM_F_FOR_ONLY|MM_F_REV_ONLY)) {
+		if ((r&1) == (q->q_pos&1)) { // forward strand
+			if (flag & MM_F_REV_ONLY) return 1;
+		} else {
+			if (flag & MM_F_FOR_ONLY) return 1;
+		}
+	}
+	return 0;
+}
+
+static mm128_t *collect_seed_hits_heap(void *km, const mm_mapopt_t *opt, int max_occ, const mm_idx_t *mi, const char *qname, const mm128_v *mv, int qlen, int64_t *n_a, int *rep_len,
+								  int *n_mini_pos, uint64_t **mini_pos)
+{
+	int i, n_m, heap_size = 0;
+	int64_t j, n_for = 0, n_rev = 0;
+	mm_match_t *m;
+	mm128_t *a, *heap;
+
+	m = collect_matches(km, &n_m, max_occ, mi, mv, n_a, rep_len, n_mini_pos, mini_pos);
+
+	heap = (mm128_t*)kmalloc(km, n_m * sizeof(mm128_t));
+	a = (mm128_t*)kmalloc(km, *n_a * sizeof(mm128_t));
+
+	for (i = 0, heap_size = 0; i < n_m; ++i) {
+		if (m[i].n > 0) {
+			heap[heap_size].x = m[i].cr[0];
+			heap[heap_size].y = (uint64_t)i<<32;
+			++heap_size;
+		}
+	}
+	ks_heapmake_heap(heap_size, heap);
+	while (heap_size > 0) {
+		mm_match_t *q = &m[heap->y>>32];
+		mm128_t *p;
+		uint64_t r = heap->x;
+		int32_t is_self, rpos = (uint32_t)r >> 1;
+		if (!skip_seed(opt->flag, r, q, qname, qlen, mi, &is_self)) {
+			if ((r&1) == (q->q_pos&1)) { // forward strand
+				p = &a[n_for++];
+				p->x = (r&0xffffffff00000000ULL) | rpos;
+				p->y = (uint64_t)q->q_span << 32 | q->q_pos >> 1;
+			} else { // reverse strand
+				p = &a[(*n_a) - (++n_rev)];
+				p->x = 1ULL<<63 | (r&0xffffffff00000000ULL) | rpos;
+				p->y = (uint64_t)q->q_span << 32 | (qlen - ((q->q_pos>>1) + 1 - q->q_span) - 1);
 			}
+			p->y |= (uint64_t)q->seg_id << MM_SEED_SEG_SHIFT;
+			if (q->is_tandem) p->y |= MM_SEED_TANDEM;
+			if (is_self) p->y |= MM_SEED_SELF;
 		}
-		return en;
-	} else return st < n && z[st] < x? st + 1 : en;
+		// update the heap
+		if ((uint32_t)heap->y < q->n - 1) {
+			++heap[0].y;
+			heap[0].x = m[heap[0].y>>32].cr[(uint32_t)heap[0].y];
+		} else {
+			heap[0] = heap[heap_size - 1];
+			--heap_size;
+		}
+		ks_heapdown_heap(0, heap_size, heap);
+	}
+	kfree(km, m);
+	kfree(km, heap);
+
+	// reverse anchors on the reverse strand, as they are in the descending order
+	for (j = 0; j < n_rev>>1; ++j) {
+		mm128_t t = a[(*n_a) - 1 - j];
+		a[(*n_a) - 1 - j] = a[(*n_a) - (n_rev - j)];
+		a[(*n_a) - (n_rev - j)] = t;
+	}
+	if (*n_a > n_for + n_rev) {
+		memmove(a + n_for, a + (*n_a) - n_rev, n_rev * sizeof(mm128_t));
+		*n_a = n_for + n_rev;
+	}
+	return a;
 }
 
-void mm_pair_thin(mm_tbuf_t *b, int radius, mm_match_t *m1, mm_match_t *m2)
+static mm128_t *collect_seed_hits(void *km, const mm_mapopt_t *opt, int max_occ, const mm_idx_t *mi, const char *qname, const mm128_v *mv, int qlen, int64_t *n_a, int *rep_len,
+								  int *n_mini_pos, uint64_t **mini_pos)
 {
-	mm_match_t *m[2];
-	const uint64_t *z[2];
-	uint64_v a[2];
-	int i, n[2], k[2], u = 0, rel = (m1->qpos ^ m2->qpos) & 1;
-
-	m[0] = m1, m[1] = m2;
-	for (i = 0; i < 2; ++i) {
-		n[i] = m[i]->n;
-		z[i] = m[i]->x.cr;
-		k[i] = 0;
-		kv_init(a[i]);
-		kv_resize(uint64_t, b->km, a[i], 256);
-	}
-	while (k[0] < n[0] && k[1] < n[1]) {
-		//printf("%d; %d,%d\n", u, k[0], k[1]);
-		int v = u^1, dist = (int)(m[v]->qpos>>1) - (int)(m[u]->qpos>>1);
-		uint64_t x = z[u][k[u]];
-		int uori = (x ^ m[u]->qpos) & 1, last;
-		int64_t tpos = x>>1 & 0x7fffffff;
-		tpos = uori == 0? tpos + dist : tpos - dist;
-		if (tpos < 0) tpos = 0;
-		x = x>>32<<32 | tpos<<1 | (x&1);
-		last = a[v].n;
-		k[v] = mm_pair_thin_core(b, x, radius, rel, k[v], n[v], z[v], &a[v]);
-		if (a[v].n > last) kv_push(uint64_t, b->km, a[u], z[u][k[u]]);
-		++k[u];
-		u ^= 1;
-	}
-	for (i = 0; i < 2; ++i)
-		m[i]->n = a[i].n, m[i]->x.r = a[i].a, m[i]->is_alloc = 1;
-//	printf("%d,%d; %d,%d\n", m[0]->qpos>>1, m[1]->qpos>>1, m[0]->n, m[1]->n);
-}
-#endif
-mm_reg1_t *mm_map_frag(const mm_mapopt_t *opt, const mm_idx_t *mi, mm_tbuf_t *b, uint32_t m_st, uint32_t m_en, const char *qname, int qlen, const char *seq, int *n_regs)
-{
-	int i, n = m_en - m_st, j, n_u, max_gap_ref;
-	int64_t n_a;
-	uint64_t *u;
+	int i, n_m;
 	mm_match_t *m;
 	mm128_t *a;
-	mm_reg1_t *regs;
-
-	// convert to local representation
-	m = (mm_match_t*)kmalloc(b->km, n * sizeof(mm_match_t));
-	for (i = 0; i < n; ++i) {
-		int t;
-		mm128_t *p = &b->mini.a[i + m_st];
-		m[i].is_alloc = 0;
-		m[i].qpos = (uint32_t)p->y;
-		m[i].x.cr = mm_idx_get(mi, p->x>>8, &t);
-		m[i].n = t;
-	}
-#if 0
-	int last = -1, last2 = -1;
-	// pair k-mer thinning
-	for (i = 0; i < n; ++i) {
-		if (m[i].n >= opt->mid_occ && m[i].n < opt->max_occ) {
-			if (last2 < 0) last2 = i;
-			if (last < 0 || m[last].n < m[i].n) last = i;
-			if (last >= 0 && (m[last].qpos>>1) + (m[last].span>>1) <= m[i].qpos>>1) {
-				mm_pair_thin(b, opt->bw, &m[last], &m[i]);
-				last2 = last = -1;
-			} else if (last2 >= 0 && (m[last2].qpos>>1) + (m[last2].span>>1) <= m[i].qpos>>1) {
-				mm_pair_thin(b, opt->bw, &m[last2], &m[i]);
-				last2 = last = -1;
-			}
-		}
-	}
-#endif
-	// fill the _a_ array
-	for (i = 0, n_a = 0; i < n; ++i) // find the length of a[]
-		if (m[i].n < opt->mid_occ) n_a += m[i].n;
-	a = (mm128_t*)kmalloc(b->km, n_a * sizeof(mm128_t));
-	for (i = j = 0; i < n; ++i) {
-		mm128_t *p = &b->mini.a[i + m_st];
+	m = collect_matches(km, &n_m, max_occ, mi, mv, n_a, rep_len, n_mini_pos, mini_pos);
+	a = (mm128_t*)kmalloc(km, *n_a * sizeof(mm128_t));
+	for (i = 0, *n_a = 0; i < n_m; ++i) {
 		mm_match_t *q = &m[i];
-		const uint64_t *r = q->x.cr;
-		int k, q_span = p->x & 0xff, is_tandem = 0;
-		if (q->n >= opt->mid_occ) continue;
-		if (i > 0 && p->x>>8 == b->mini.a[m_st + i - 1].x>>8) is_tandem = 1;
-		if (i < n - 1 && p->x>>8 == b->mini.a[m_st + i + 1].x>>8) is_tandem = 1;
+		const uint64_t *r = q->cr;
+		uint32_t k;
 		for (k = 0; k < q->n; ++k) {
-			const char *tname = mi->seq[r[k]>>32].name;
-			int32_t rpos = (uint32_t)r[k] >> 1;
+			int32_t is_self, rpos = (uint32_t)r[k] >> 1;
 			mm128_t *p;
-			if (qname && (opt->flag&MM_F_NO_SELF) && strcmp(qname, tname) == 0 && rpos == (q->qpos>>1)) // avoid the diagonal
-				continue;
-			if (qname && (opt->flag&MM_F_AVA) && strcmp(qname, tname) > 0) // all-vs-all mode: map once
-				continue;
-			p = &a[j++];
-			if ((r[k]&1) == (q->qpos&1)) { // forward strand
-				p->x = (r[k]&0xffffffff00000000ULL) | (uint32_t)r[k]>>1;
-				p->y = (uint64_t)q_span << 32 | q->qpos >> 1;
+			if (skip_seed(opt->flag, r[k], q, qname, qlen, mi, &is_self)) continue;
+			p = &a[(*n_a)++];
+			if ((r[k]&1) == (q->q_pos&1)) { // forward strand
+				p->x = (r[k]&0xffffffff00000000ULL) | rpos;
+				p->y = (uint64_t)q->q_span << 32 | q->q_pos >> 1;
 			} else { // reverse strand
-				p->x = 1ULL<<63 | (r[k]&0xffffffff00000000ULL) | (uint32_t)r[k]>>1;
-				p->y = (uint64_t)q_span << 32 | (qlen - ((q->qpos>>1) + 1 - q_span) - 1);
+				p->x = 1ULL<<63 | (r[k]&0xffffffff00000000ULL) | rpos;
+				p->y = (uint64_t)q->q_span << 32 | (qlen - ((q->q_pos>>1) + 1 - q->q_span) - 1);
 			}
-			if (is_tandem) p->y |= MM_SEED_TANDEM;
+			p->y |= (uint64_t)q->seg_id << MM_SEED_SEG_SHIFT;
+			if (q->is_tandem) p->y |= MM_SEED_TANDEM;
+			if (is_self) p->y |= MM_SEED_SELF;
 		}
 	}
-	n_a = j;
-	radix_sort_128x(a, a + n_a);
-	for (i = 0; i < n; ++i)
-		if (m[i].is_alloc) kfree(b->km, m[i].x.r);
-	kfree(b->km, m);
+	kfree(km, m);
+	radix_sort_128x(a, a + (*n_a));
+	return a;
+}
 
-	if (mm_dbg_flag & MM_DBG_PRINT_SEED)
-		for (i = 0; i < n_a; ++i)
-			fprintf(stderr, "SD\t%s\t%d\t%c\t%d\t%d\t%d\n", mi->seq[a[i].x<<1>>33].name, (int32_t)a[i].x, "+-"[a[i].x>>63], (int32_t)a[i].y, (int32_t)(a[i].y>>32&0xff),
-					i == 0? 0 : ((int32_t)a[i].y - (int32_t)a[i-1].y) - ((int32_t)a[i].x - (int32_t)a[i-1].x));
-
-	max_gap_ref = opt->max_gap_ref >= 0? opt->max_gap_ref : opt->max_gap;
-	n_u = mm_chain_dp(max_gap_ref, opt->max_gap, opt->bw, opt->max_chain_skip, opt->min_cnt, opt->min_chain_score, !!(opt->flag&MM_F_SPLICE), n_a, a, &u, b->km);
-	regs = mm_gen_regs(b->km, qlen, n_u, u, a);
-	*n_regs = n_u;
-
-	if (mm_dbg_flag & MM_DBG_PRINT_SEED)
-		for (j = 0; j < n_u; ++j)
-			for (i = regs[j].as; i < regs[j].as + regs[j].cnt; ++i)
-				fprintf(stderr, "CN\t%d\t%s\t%d\t%c\t%d\t%d\t%d\n", j, mi->seq[a[i].x<<1>>33].name, (int32_t)a[i].x, "+-"[a[i].x>>63], (int32_t)a[i].y, (int32_t)(a[i].y>>32&0xff),
-						i == regs[j].as? 0 : ((int32_t)a[i].y - (int32_t)a[i-1].y) - ((int32_t)a[i].x - (int32_t)a[i-1].x));
-
-	if (!(opt->flag & MM_F_AVA)) { // don't choose primary mapping(s) for read overlap
-		mm_set_parent(b->km, opt->mask_level, *n_regs, regs);
-		mm_select_sub(b->km, opt->mask_level, opt->pri_ratio, mi->k*2, opt->best_n, n_regs, regs);
-		if (!(opt->flag & MM_F_SPLICE))
-			mm_join_long(b->km, opt, qlen, n_regs, regs, a); // TODO: this can be applied to all-vs-all in principle
+static void chain_post(const mm_mapopt_t *opt, int max_chain_gap_ref, const mm_idx_t *mi, void *km, int qlen, int n_segs, const int *qlens, int *n_regs, mm_reg1_t *regs, mm128_t *a)
+{
+	if (!(opt->flag & MM_F_ALL_CHAINS)) { // don't choose primary mapping(s)
+		mm_set_parent(km, opt->mask_level, *n_regs, regs, opt->a * 2 + opt->b, opt->flag&MM_F_HARD_MLEVEL);
+		if (n_segs <= 1) mm_select_sub(km, opt->pri_ratio, mi->k*2, opt->best_n, n_regs, regs);
+		else mm_select_sub_multi(km, opt->pri_ratio, 0.2f, 0.7f, max_chain_gap_ref, mi->k*2, opt->best_n, n_segs, qlens, n_regs, regs);
+		if (!(opt->flag & (MM_F_SPLICE|MM_F_SR|MM_F_NO_LJOIN))) // long join not working well without primary chains
+			mm_join_long(km, opt, qlen, n_regs, regs, a);
 	}
-	if (opt->flag & MM_F_CIGAR) {
-		regs = mm_align_skeleton(b->km, opt, mi, qlen, seq, n_regs, regs, a); // this calls mm_filter_regs()
-		if (!(opt->flag & MM_F_AVA)) {
-			mm_set_parent(b->km, opt->mask_level, *n_regs, regs);
-			mm_select_sub(b->km, opt->mask_level, opt->pri_ratio, mi->k*2, opt->best_n, n_regs, regs);
-			mm_set_sam_pri(*n_regs, regs);
-		}
-	}
-	mm_set_mapq(*n_regs, regs, opt->min_chain_score);
+}
 
-	// free
-	kfree(b->km, a);
-	kfree(b->km, u);
+static mm_reg1_t *align_regs(const mm_mapopt_t *opt, const mm_idx_t *mi, void *km, int qlen, const char *seq, int *n_regs, mm_reg1_t *regs, mm128_t *a)
+{
+	if (!(opt->flag & MM_F_CIGAR)) return regs;
+	regs = mm_align_skeleton(km, opt, mi, qlen, seq, n_regs, regs, a); // this calls mm_filter_regs()
+	if (!(opt->flag & MM_F_ALL_CHAINS)) { // don't choose primary mapping(s)
+		mm_set_parent(km, opt->mask_level, *n_regs, regs, opt->a * 2 + opt->b, opt->flag&MM_F_HARD_MLEVEL);
+		mm_select_sub(km, opt->pri_ratio, mi->k*2, opt->best_n, n_regs, regs);
+		mm_set_sam_pri(*n_regs, regs);
+	}
 	return regs;
 }
 
-mm_reg1_t *mm_map(const mm_idx_t *mi, int l_seq, const char *seq, int *n_regs, mm_tbuf_t *b, const mm_mapopt_t *opt, const char *qname)
+void mm_map_frag(const mm_idx_t *mi, int n_segs, const int *qlens, const char **seqs, int *n_regs, mm_reg1_t **regs, mm_tbuf_t *b, const mm_mapopt_t *opt, const char *qname)
+{
+	int i, j, rep_len, qlen_sum, n_regs0, n_mini_pos;
+	int max_chain_gap_qry, max_chain_gap_ref, is_splice = !!(opt->flag & MM_F_SPLICE), is_sr = !!(opt->flag & MM_F_SR);
+	uint32_t hash;
+	int64_t n_a;
+	uint64_t *u, *mini_pos;
+	mm128_t *a;
+	mm128_v mv = {0,0,0};
+	mm_reg1_t *regs0;
+	km_stat_t kmst;
+
+	for (i = 0, qlen_sum = 0; i < n_segs; ++i)
+		qlen_sum += qlens[i], n_regs[i] = 0, regs[i] = 0;
+
+	if (qlen_sum == 0 || n_segs <= 0 || n_segs > MM_MAX_SEG) return;
+
+	hash  = qname? __ac_X31_hash_string(qname) : 0;
+	hash ^= __ac_Wang_hash(qlen_sum) + __ac_Wang_hash(opt->seed);
+	hash  = __ac_Wang_hash(hash);
+
+	collect_minimizers(b->km, opt, mi, n_segs, qlens, seqs, &mv);
+	if (opt->flag & MM_F_HEAP_SORT) a = collect_seed_hits_heap(b->km, opt, opt->mid_occ, mi, qname, &mv, qlen_sum, &n_a, &rep_len, &n_mini_pos, &mini_pos);
+	else a = collect_seed_hits(b->km, opt, opt->mid_occ, mi, qname, &mv, qlen_sum, &n_a, &rep_len, &n_mini_pos, &mini_pos);
+
+	if (mm_dbg_flag & MM_DBG_PRINT_SEED) {
+		fprintf(stderr, "RS\t%d\n", rep_len);
+		for (i = 0; i < n_a; ++i)
+			fprintf(stderr, "SD\t%s\t%d\t%c\t%d\t%d\t%d\n", mi->seq[a[i].x<<1>>33].name, (int32_t)a[i].x, "+-"[a[i].x>>63], (int32_t)a[i].y, (int32_t)(a[i].y>>32&0xff),
+					i == 0? 0 : ((int32_t)a[i].y - (int32_t)a[i-1].y) - ((int32_t)a[i].x - (int32_t)a[i-1].x));
+	}
+
+	// set max chaining gap on the query and the reference sequence
+	if (is_sr)
+		max_chain_gap_qry = qlen_sum > opt->max_gap? qlen_sum : opt->max_gap;
+	else max_chain_gap_qry = opt->max_gap;
+	if (opt->max_gap_ref > 0) {
+		max_chain_gap_ref = opt->max_gap_ref; // always honor mm_mapopt_t::max_gap_ref if set
+	} else if (opt->max_frag_len > 0) {
+		max_chain_gap_ref = opt->max_frag_len - qlen_sum;
+		if (max_chain_gap_ref < opt->max_gap) max_chain_gap_ref = opt->max_gap;
+	} else max_chain_gap_ref = opt->max_gap;
+
+	a = mm_chain_dp(max_chain_gap_ref, max_chain_gap_qry, opt->bw, opt->max_chain_skip, opt->min_cnt, opt->min_chain_score, is_splice, n_segs, n_a, a, &n_regs0, &u, b->km);
+
+	if (opt->max_occ > opt->mid_occ && rep_len > 0) {
+		int rechain = 0;
+		if (n_regs0 > 0) { // test if the best chain has all the segments
+			int n_chained_segs = 1, max = 0, max_i = -1, max_off = -1, off = 0;
+			for (i = 0; i < n_regs0; ++i) { // find the best chain
+				if (max < (int)(u[i]>>32)) max = u[i]>>32, max_i = i, max_off = off;
+				off += (uint32_t)u[i];
+			}
+			for (i = 1; i < (int32_t)u[max_i]; ++i) // count the number of segments in the best chain
+				if ((a[max_off+i].y&MM_SEED_SEG_MASK) != (a[max_off+i-1].y&MM_SEED_SEG_MASK))
+					++n_chained_segs;
+			if (n_chained_segs < n_segs)
+				rechain = 1;
+		} else rechain = 1;
+		if (rechain) { // redo chaining with a higher max_occ threshold
+			kfree(b->km, a);
+			kfree(b->km, u);
+			kfree(b->km, mini_pos);
+			if (opt->flag & MM_F_HEAP_SORT) a = collect_seed_hits_heap(b->km, opt, opt->max_occ, mi, qname, &mv, qlen_sum, &n_a, &rep_len, &n_mini_pos, &mini_pos);
+			else a = collect_seed_hits(b->km, opt, opt->max_occ, mi, qname, &mv, qlen_sum, &n_a, &rep_len, &n_mini_pos, &mini_pos);
+			a = mm_chain_dp(max_chain_gap_ref, max_chain_gap_qry, opt->bw, opt->max_chain_skip, opt->min_cnt, opt->min_chain_score, is_splice, n_segs, n_a, a, &n_regs0, &u, b->km);
+		}
+	}
+	b->frag_gap = max_chain_gap_ref;
+	b->rep_len = rep_len;
+
+	regs0 = mm_gen_regs(b->km, hash, qlen_sum, n_regs0, u, a);
+
+	if (mm_dbg_flag & MM_DBG_PRINT_SEED)
+		for (j = 0; j < n_regs0; ++j)
+			for (i = regs0[j].as; i < regs0[j].as + regs0[j].cnt; ++i)
+				fprintf(stderr, "CN\t%d\t%s\t%d\t%c\t%d\t%d\t%d\n", j, mi->seq[a[i].x<<1>>33].name, (int32_t)a[i].x, "+-"[a[i].x>>63], (int32_t)a[i].y, (int32_t)(a[i].y>>32&0xff),
+						i == regs0[j].as? 0 : ((int32_t)a[i].y - (int32_t)a[i-1].y) - ((int32_t)a[i].x - (int32_t)a[i-1].x));
+
+	chain_post(opt, max_chain_gap_ref, mi, b->km, qlen_sum, n_segs, qlens, &n_regs0, regs0, a);
+	if (!is_sr) mm_est_err(mi, qlen_sum, n_regs0, regs0, a, n_mini_pos, mini_pos);
+
+	if (n_segs == 1) { // uni-segment
+		regs0 = align_regs(opt, mi, b->km, qlens[0], seqs[0], &n_regs0, regs0, a);
+		mm_set_mapq(b->km, n_regs0, regs0, opt->min_chain_score, opt->a, rep_len, is_sr);
+		n_regs[0] = n_regs0, regs[0] = regs0;
+	} else { // multi-segment
+		mm_seg_t *seg;
+		seg = mm_seg_gen(b->km, hash, n_segs, qlens, n_regs0, regs0, n_regs, regs, a); // split fragment chain to separate segment chains
+		free(regs0);
+		for (i = 0; i < n_segs; ++i) {
+			mm_set_parent(b->km, opt->mask_level, n_regs[i], regs[i], opt->a * 2 + opt->b, opt->flag&MM_F_HARD_MLEVEL); // update mm_reg1_t::parent
+			regs[i] = align_regs(opt, mi, b->km, qlens[i], seqs[i], &n_regs[i], regs[i], seg[i].a);
+			mm_set_mapq(b->km, n_regs[i], regs[i], opt->min_chain_score, opt->a, rep_len, is_sr);
+		}
+		mm_seg_free(b->km, n_segs, seg);
+		if (n_segs == 2 && opt->pe_ori >= 0 && (opt->flag&MM_F_CIGAR))
+			mm_pair(b->km, max_chain_gap_ref, opt->pe_bonus, opt->a * 2 + opt->b, opt->a, qlens, n_regs, regs); // pairing
+	}
+
+	kfree(b->km, mv.a);
+	kfree(b->km, a);
+	kfree(b->km, u);
+	kfree(b->km, mini_pos);
+
+	if (b->km) {
+		km_stat(b->km, &kmst);
+		if (mm_dbg_flag & MM_DBG_PRINT_QNAME)
+			fprintf(stderr, "QM\t%s\t%d\tcap=%ld,nCore=%ld,largest=%ld\n", qname, qlen_sum, kmst.capacity, kmst.n_cores, kmst.largest);
+		assert(kmst.n_blocks == kmst.n_cores); // otherwise, there is a memory leak
+		if (kmst.largest > 1U<<28) {
+			km_destroy(b->km);
+			b->km = km_init();
+		}
+	}
+}
+
+mm_reg1_t *mm_map(const mm_idx_t *mi, int qlen, const char *seq, int *n_regs, mm_tbuf_t *b, const mm_mapopt_t *opt, const char *qname)
 {
 	mm_reg1_t *regs;
-	b->mini.n = 0;
-	mm_sketch(b->km, seq, l_seq, mi->w, mi->k, 0, mi->is_hpc, &b->mini);
-	if (opt->sdust_thres > 0)
-		mm_dust_minier(&b->mini, l_seq, seq, opt->sdust_thres, b->sdb);
-	regs = mm_map_frag(opt, mi, b, 0, b->mini.n, qname, l_seq, seq, n_regs);
+	mm_map_frag(mi, 1, &qlen, &seq, n_regs, &regs, b, opt, qname);
 	return regs;
 }
 
@@ -295,39 +397,137 @@ mm_reg1_t *mm_map(const mm_idx_t *mi, int l_seq, const char *seq, int *n_regs, m
  **************************/
 
 typedef struct {
-	int mini_batch_size, n_processed, n_threads;
+	int mini_batch_size, n_processed, n_threads, n_fp;
 	const mm_mapopt_t *opt;
-	mm_bseq_file_t *fp;
+	mm_bseq_file_t **fp;
 	const mm_idx_t *mi;
 	kstring_t str;
+
+	int n_parts;
+	uint32_t *rid_shift;
+	FILE *fp_split, **fp_parts;
 } pipeline_t;
 
 typedef struct {
 	const pipeline_t *p;
-    int n_seq;
+    int n_seq, n_frag;
 	mm_bseq1_t *seq;
-	int *n_reg;
+	int *n_reg, *seg_off, *n_seg, *rep_len, *frag_gap;
 	mm_reg1_t **reg;
 	mm_tbuf_t **buf;
 } step_t;
 
 static void worker_for(void *_data, long i, int tid) // kt_for() callback
 {
-    step_t *step = (step_t*)_data;
+    step_t *s = (step_t*)_data;
+	int qlens[MM_MAX_SEG], j, off = s->seg_off[i], pe_ori = s->p->opt->pe_ori;
+	const char *qseqs[MM_MAX_SEG];
+	mm_tbuf_t *b = s->buf[tid];
+	assert(s->n_seg[i] <= MM_MAX_SEG);
 	if (mm_dbg_flag & MM_DBG_PRINT_QNAME)
-		fprintf(stderr, "QR\t%s\t%d\n", step->seq[i].name, tid);
-	step->reg[i] = mm_map(step->p->mi, step->seq[i].l_seq, step->seq[i].seq, &step->n_reg[i], step->buf[tid], step->p->opt, step->seq[i].name);
+		fprintf(stderr, "QR\t%s\t%d\t%d\n", s->seq[off].name, tid, s->seq[off].l_seq);
+	for (j = 0; j < s->n_seg[i]; ++j) {
+		if (s->n_seg[i] == 2 && ((j == 0 && (pe_ori>>1&1)) || (j == 1 && (pe_ori&1))))
+			mm_revcomp_bseq(&s->seq[off + j]);
+		qlens[j] = s->seq[off + j].l_seq;
+		qseqs[j] = s->seq[off + j].seq;
+	}
+	if (s->p->opt->flag & MM_F_INDEPEND_SEG) {
+		for (j = 0; j < s->n_seg[i]; ++j) {
+			mm_map_frag(s->p->mi, 1, &qlens[j], &qseqs[j], &s->n_reg[off+j], &s->reg[off+j], b, s->p->opt, s->seq[off+j].name);
+			s->rep_len[off + j] = b->rep_len;
+			s->frag_gap[off + j] = b->frag_gap;
+		}
+	} else {
+		mm_map_frag(s->p->mi, s->n_seg[i], qlens, qseqs, &s->n_reg[off], &s->reg[off], b, s->p->opt, s->seq[off].name);
+		for (j = 0; j < s->n_seg[i]; ++j) {
+			s->rep_len[off + j] = b->rep_len;
+			s->frag_gap[off + j] = b->frag_gap;
+		}
+	}
+	for (j = 0; j < s->n_seg[i]; ++j) // flip the query strand and coordinate to the original read strand
+		if (s->n_seg[i] == 2 && ((j == 0 && (pe_ori>>1&1)) || (j == 1 && (pe_ori&1)))) {
+			int k, t;
+			mm_revcomp_bseq(&s->seq[off + j]);
+			for (k = 0; k < s->n_reg[off + j]; ++k) {
+				mm_reg1_t *r = &s->reg[off + j][k];
+				t = r->qs;
+				r->qs = qlens[j] - r->qe;
+				r->qe = qlens[j] - t;
+				r->rev = !r->rev;
+			}
+		}
+}
+
+static void merge_hits(step_t *s)
+{
+	int f, i, k0, k, max_seg = 0, *n_reg_part, *rep_len_part, *frag_gap_part, *qlens;
+	void *km;
+	FILE **fp = s->p->fp_parts;
+	const mm_mapopt_t *opt = s->p->opt;
+
+	km = km_init();
+	for (f = 0; f < s->n_frag; ++f)
+		max_seg = max_seg > s->n_seg[f]? max_seg : s->n_seg[f];
+	qlens = CALLOC(int, max_seg + s->p->n_parts * 3);
+	n_reg_part = qlens + max_seg;
+	rep_len_part = n_reg_part + s->p->n_parts;
+	frag_gap_part = rep_len_part + s->p->n_parts;
+	for (f = 0, k = k0 = 0; f < s->n_frag; ++f) {
+		k0 = k;
+		for (i = 0; i < s->n_seg[f]; ++i, ++k) {
+			int j, l, t, rep_len = 0;
+			qlens[i] = s->seq[k].l_seq;
+			for (j = 0, s->n_reg[k] = 0; j < s->p->n_parts; ++j) {
+				mm_err_fread(&n_reg_part[j],    sizeof(int), 1, fp[j]);
+				mm_err_fread(&rep_len_part[j],  sizeof(int), 1, fp[j]);
+				mm_err_fread(&frag_gap_part[j], sizeof(int), 1, fp[j]);
+				s->n_reg[k] += n_reg_part[j];
+				if (rep_len < rep_len_part[j])
+					rep_len = rep_len_part[j];
+			}
+			s->reg[k] = CALLOC(mm_reg1_t, s->n_reg[k]);
+			for (j = 0, l = 0; j < s->p->n_parts; ++j) {
+				for (t = 0; t < n_reg_part[j]; ++t, ++l) {
+					mm_reg1_t *r = &s->reg[k][l];
+					uint32_t capacity;
+					mm_err_fread(r, sizeof(mm_reg1_t), 1, fp[j]);
+					r->rid += s->p->rid_shift[j];
+					if (opt->flag & MM_F_CIGAR) {
+						mm_err_fread(&capacity, 4, 1, fp[j]);
+						r->p = (mm_extra_t*)calloc(capacity, 4);
+						r->p->capacity = capacity;
+						mm_err_fread(r->p, r->p->capacity, 4, fp[j]);
+					}
+				}
+			}
+			mm_hit_sort(km, &s->n_reg[k], s->reg[k]);
+			mm_set_parent(km, opt->mask_level, s->n_reg[k], s->reg[k], opt->a * 2 + opt->b, opt->flag&MM_F_HARD_MLEVEL);
+			if (!(opt->flag & MM_F_ALL_CHAINS)) {
+				mm_select_sub(km, opt->pri_ratio, s->p->mi->k*2, opt->best_n, &s->n_reg[k], s->reg[k]);
+				mm_set_sam_pri(s->n_reg[k], s->reg[k]);
+			}
+			mm_set_mapq(km, s->n_reg[k], s->reg[k], opt->min_chain_score, opt->a, rep_len, !!(opt->flag & MM_F_SR));
+		}
+		if (s->n_seg[f] == 2 && opt->pe_ori >= 0 && (opt->flag&MM_F_CIGAR))
+			mm_pair(km, frag_gap_part[0], opt->pe_bonus, opt->a * 2 + opt->b, opt->a, qlens, &s->n_reg[k0], &s->reg[k0]);
+	}
+	free(qlens);
+	km_destroy(km);
 }
 
 static void *worker_pipeline(void *shared, int step, void *in)
 {
-	int i, j;
+	int i, j, k;
     pipeline_t *p = (pipeline_t*)shared;
     if (step == 0) { // step 0: read sequences
 		int with_qual = (!!(p->opt->flag & MM_F_OUT_SAM) && !(p->opt->flag & MM_F_NO_QUAL));
+		int with_comment = !!(p->opt->flag & MM_F_COPY_COMMENT);
+		int frag_mode = (p->n_fp > 1 || !!(p->opt->flag & MM_F_FRAG_MODE));
         step_t *s;
         s = (step_t*)calloc(1, sizeof(step_t));
-		s->seq = mm_bseq_read(p->fp, p->mini_batch_size, with_qual, &s->n_seq);
+		if (p->n_fp > 1) s->seq = mm_bseq_read_frag2(p->n_fp, p->fp, p->mini_batch_size, with_qual, with_comment, &s->n_seq);
+		else s->seq = mm_bseq_read3(p->fp[0], p->mini_batch_size, with_qual, with_comment, frag_mode, &s->n_seq);
 		if (s->seq) {
 			s->p = p;
 			for (i = 0; i < s->n_seq; ++i)
@@ -335,12 +535,23 @@ static void *worker_pipeline(void *shared, int step, void *in)
 			s->buf = (mm_tbuf_t**)calloc(p->n_threads, sizeof(mm_tbuf_t*));
 			for (i = 0; i < p->n_threads; ++i)
 				s->buf[i] = mm_tbuf_init();
-			s->n_reg = (int*)calloc(s->n_seq, sizeof(int));
+			s->n_reg = (int*)calloc(5 * s->n_seq, sizeof(int));
+			s->seg_off = s->n_reg + s->n_seq; // seg_off, n_seg, rep_len and frag_gap are allocated together with n_reg
+			s->n_seg = s->seg_off + s->n_seq;
+			s->rep_len = s->n_seg + s->n_seq;
+			s->frag_gap = s->rep_len + s->n_seq;
 			s->reg = (mm_reg1_t**)calloc(s->n_seq, sizeof(mm_reg1_t*));
+			for (i = 1, j = 0; i <= s->n_seq; ++i)
+				if (i == s->n_seq || !frag_mode || !mm_qname_same(s->seq[i-1].name, s->seq[i].name)) {
+					s->n_seg[s->n_frag] = i - j;
+					s->seg_off[s->n_frag++] = j;
+					j = i;
+				}
 			return s;
 		} else free(s);
     } else if (step == 1) { // step 1: map
-		kt_for(p->n_threads, worker_for, in, ((step_t*)in)->n_seq);
+		if (p->n_parts > 0) merge_hits((step_t*)in);
+		else kt_for(p->n_threads, worker_for, in, ((step_t*)in)->n_frag);
 		return in;
     } else if (step == 2) { // step 2: output
 		void *km = 0;
@@ -349,26 +560,51 @@ static void *worker_pipeline(void *shared, int step, void *in)
 		for (i = 0; i < p->n_threads; ++i) mm_tbuf_destroy(s->buf[i]);
 		free(s->buf);
 		if ((p->opt->flag & MM_F_OUT_CS) && !(mm_dbg_flag & MM_DBG_NO_KALLOC)) km = km_init();
-		for (i = 0; i < s->n_seq; ++i) {
-			mm_bseq1_t *t = &s->seq[i];
-			for (j = 0; j < s->n_reg[i]; ++j) {
-				mm_reg1_t *r = &s->reg[i][j];
-				if (p->opt->flag & MM_F_OUT_SAM)
-					mm_write_sam(&p->str, mi, t, r, s->n_reg[i], s->reg[i]);
-				else
-					mm_write_paf(&p->str, mi, t, r, km, p->opt->flag);
-				puts(p->str.s);
+		for (k = 0; k < s->n_frag; ++k) {
+			int seg_st = s->seg_off[k], seg_en = s->seg_off[k] + s->n_seg[k];
+			for (i = seg_st; i < seg_en; ++i) {
+				mm_bseq1_t *t = &s->seq[i];
+				if (p->opt->split_prefix && p->n_parts == 0) { // then write to temporary files
+					mm_err_fwrite(&s->n_reg[i],    sizeof(int), 1, p->fp_split);
+					mm_err_fwrite(&s->rep_len[i],  sizeof(int), 1, p->fp_split);
+					mm_err_fwrite(&s->frag_gap[i], sizeof(int), 1, p->fp_split);
+					for (j = 0; j < s->n_reg[i]; ++j) {
+						mm_reg1_t *r = &s->reg[i][j];
+						mm_err_fwrite(r, sizeof(mm_reg1_t), 1, p->fp_split);
+						if (p->opt->flag & MM_F_CIGAR) {
+							mm_err_fwrite(&r->p->capacity, 4, 1, p->fp_split);
+							mm_err_fwrite(r->p, r->p->capacity, 4, p->fp_split);
+						}
+					}
+				} else if (s->n_reg[i] > 0) { // the query has at least one hit
+					for (j = 0; j < s->n_reg[i]; ++j) {
+						mm_reg1_t *r = &s->reg[i][j];
+						assert(!r->sam_pri || r->id == r->parent);
+						if ((p->opt->flag & MM_F_NO_PRINT_2ND) && r->id != r->parent)
+							continue;
+						if (p->opt->flag & MM_F_OUT_SAM)
+							mm_write_sam2(&p->str, mi, t, i - seg_st, j, s->n_seg[k], &s->n_reg[seg_st], (const mm_reg1_t*const*)&s->reg[seg_st], km, p->opt->flag);
+						else
+							mm_write_paf(&p->str, mi, t, r, km, p->opt->flag);
+						mm_err_puts(p->str.s);
+					}
+				} else if (p->opt->flag & (MM_F_OUT_SAM|MM_F_PAF_NO_HIT)) { // output an empty hit, if requested
+					if (p->opt->flag & MM_F_OUT_SAM)
+						mm_write_sam2(&p->str, mi, t, i - seg_st, -1, s->n_seg[k], &s->n_reg[seg_st], (const mm_reg1_t*const*)&s->reg[seg_st], km, p->opt->flag);
+					else
+						mm_write_paf(&p->str, mi, t, 0, 0, p->opt->flag);
+					mm_err_puts(p->str.s);
+				}
 			}
-			if (s->n_reg[i] == 0 && (p->opt->flag & MM_F_OUT_SAM)) {
-				mm_write_sam(&p->str, 0, t, 0, 0, 0);
-				puts(p->str.s);
+			for (i = seg_st; i < seg_en; ++i) {
+				for (j = 0; j < s->n_reg[i]; ++j) free(s->reg[i][j].p);
+				free(s->reg[i]);
+				free(s->seq[i].seq); free(s->seq[i].name);
+				if (s->seq[i].qual) free(s->seq[i].qual);
+				if (s->seq[i].comment) free(s->seq[i].comment);
 			}
-			for (j = 0; j < s->n_reg[i]; ++j) free(s->reg[i][j].p);
-			free(s->reg[i]);
-			free(s->seq[i].seq); free(s->seq[i].name);
-			if (s->seq[i].qual) free(s->seq[i].qual);
 		}
-		free(s->reg); free(s->n_reg); free(s->seq);
+		free(s->reg); free(s->n_reg); free(s->seq); // seg_off, n_seg, rep_len and frag_gap were allocated with reg; no memory leak here
 		km_destroy(km);
 		if (mm_verbose >= 3)
 			fprintf(stderr, "[M::%s::%.3f*%.2f] mapped %d sequences\n", __func__, realtime() - mm_realtime0, cputime() / (realtime() - mm_realtime0), s->n_seq);
@@ -377,18 +613,95 @@ static void *worker_pipeline(void *shared, int step, void *in)
     return 0;
 }
 
-int mm_map_file(const mm_idx_t *idx, const char *fn, const mm_mapopt_t *opt, int n_threads, int mini_batch_size)
+static mm_bseq_file_t **open_bseqs(int n, const char **fn)
 {
+	mm_bseq_file_t **fp;
+	int i, j;
+	fp = (mm_bseq_file_t**)calloc(n, sizeof(mm_bseq_file_t*));
+	for (i = 0; i < n; ++i) {
+		if ((fp[i] = mm_bseq_open(fn[i])) == 0) {
+			if (mm_verbose >= 1)
+				fprintf(stderr, "ERROR: failed to open file '%s'\n", fn[i]);
+			for (j = 0; j < i; ++j)
+				mm_bseq_close(fp[j]);
+			free(fp);
+			return 0;
+		}
+	}
+	return fp;
+}
+
+int mm_map_file_frag(const mm_idx_t *idx, int n_segs, const char **fn, const mm_mapopt_t *opt, int n_threads)
+{
+	int i, pl_threads;
 	pipeline_t pl;
+	if (n_segs < 1) return -1;
 	memset(&pl, 0, sizeof(pipeline_t));
-	pl.fp = mm_bseq_open(fn);
+	pl.n_fp = n_segs;
+	pl.fp = open_bseqs(pl.n_fp, fn);
 	if (pl.fp == 0) return -1;
 	pl.opt = opt, pl.mi = idx;
-	pl.n_threads = n_threads, pl.mini_batch_size = mini_batch_size;
-	if ((opt->flag & MM_F_OUT_SAM) && !(opt->flag & MM_F_NO_SAM_SQ))
-		mm_write_sam_SQ(idx);
-	kt_pipeline(n_threads == 1? 1 : 2, worker_pipeline, &pl, 3);
+	pl.n_threads = n_threads > 1? n_threads : 1;
+	pl.mini_batch_size = opt->mini_batch_size;
+	if (opt->split_prefix)
+		pl.fp_split = mm_split_init(opt->split_prefix, idx);
+	pl_threads = n_threads == 1? 1 : (opt->flag&MM_F_2_IO_THREADS)? 3 : 2;
+	kt_pipeline(pl_threads, worker_pipeline, &pl, 3);
+
 	free(pl.str.s);
-	mm_bseq_close(pl.fp);
+	if (pl.fp_split) fclose(pl.fp_split);
+	for (i = 0; i < pl.n_fp; ++i)
+		mm_bseq_close(pl.fp[i]);
+	free(pl.fp);
+	return 0;
+}
+
+int mm_map_file(const mm_idx_t *idx, const char *fn, const mm_mapopt_t *opt, int n_threads)
+{
+	return mm_map_file_frag(idx, 1, &fn, opt, n_threads);
+}
+
+int mm_split_merge(int n_segs, const char **fn, const mm_mapopt_t *opt, int n_split_idx)
+{
+	int i;
+	pipeline_t pl;
+	mm_idx_t *mi;
+	if (n_segs < 1 || n_split_idx < 1) return -1;
+	memset(&pl, 0, sizeof(pipeline_t));
+	pl.n_fp = n_segs;
+	pl.fp = open_bseqs(pl.n_fp, fn);
+	if (pl.fp == 0) return -1;
+	pl.opt = opt;
+	pl.mini_batch_size = opt->mini_batch_size;
+
+	pl.n_parts = n_split_idx;
+	pl.fp_parts  = CALLOC(FILE*, pl.n_parts);
+	pl.rid_shift = CALLOC(uint32_t, pl.n_parts);
+	pl.mi = mi = mm_split_merge_prep(opt->split_prefix, n_split_idx, pl.fp_parts, pl.rid_shift);
+	if (pl.mi == 0) {
+		free(pl.fp_parts);
+		free(pl.rid_shift);
+		return -1;
+	}
+	for (i = n_split_idx - 1; i > 0; --i)
+		pl.rid_shift[i] = pl.rid_shift[i - 1];
+	for (pl.rid_shift[0] = 0, i = 1; i < n_split_idx; ++i)
+		pl.rid_shift[i] += pl.rid_shift[i - 1];
+	if (opt->flag & MM_F_OUT_SAM)
+		for (i = 0; i < (int32_t)pl.mi->n_seq; ++i)
+			printf("@SQ\tSN:%s\tLN:%d\n", pl.mi->seq[i].name, pl.mi->seq[i].len);
+
+	kt_pipeline(2, worker_pipeline, &pl, 3);
+
+	free(pl.str.s);
+	mm_idx_destroy(mi);
+	free(pl.rid_shift);
+	for (i = 0; i < n_split_idx; ++i)
+		fclose(pl.fp_parts[i]);
+	free(pl.fp_parts);
+	for (i = 0; i < pl.n_fp; ++i)
+		mm_bseq_close(pl.fp[i]);
+	free(pl.fp);
+	mm_split_rm_tmp(opt->split_prefix, n_split_idx);
 	return 0;
 }

minimap.h

@@ -5,42 +5,54 @@
 #include <stdio.h>
 #include <sys/types.h>
 
-#define MM_IDX_DEF_B     14
+#define MM_F_NO_DIAG       0x001 // no exact diagonal hit
+#define MM_F_NO_DUAL       0x002 // skip pairs where query name is lexicographically larger than target name
+#define MM_F_CIGAR         0x004
+#define MM_F_OUT_SAM       0x008
+#define MM_F_NO_QUAL       0x010
+#define MM_F_OUT_CG        0x020
+#define MM_F_OUT_CS        0x040
+#define MM_F_SPLICE        0x080 // splice mode
+#define MM_F_SPLICE_FOR    0x100 // match GT-AG
+#define MM_F_SPLICE_REV    0x200 // match CT-AC, the reverse complement of GT-AG
+#define MM_F_NO_LJOIN      0x400
+#define MM_F_OUT_CS_LONG   0x800
+#define MM_F_SR            0x1000
+#define MM_F_FRAG_MODE     0x2000
+#define MM_F_NO_PRINT_2ND  0x4000
+#define MM_F_2_IO_THREADS  0x8000
+#define MM_F_LONG_CIGAR    0x10000
+#define MM_F_INDEPEND_SEG  0x20000
+#define MM_F_SPLICE_FLANK  0x40000
+#define MM_F_SOFTCLIP      0x80000
+#define MM_F_FOR_ONLY      0x100000
+#define MM_F_REV_ONLY      0x200000
+#define MM_F_HEAP_SORT     0x400000
+#define MM_F_ALL_CHAINS    0x800000
+#define MM_F_OUT_MD        0x1000000
+#define MM_F_COPY_COMMENT  0x2000000
+#define MM_F_EQX           0x4000000 // use =/X instead of M
+#define MM_F_PAF_NO_HIT    0x8000000 // output unmapped reads to PAF
+#define MM_F_NO_END_FLT    0x10000000
+#define MM_F_HARD_MLEVEL   0x20000000
 
-#define MM_F_NO_SELF     0x001
-#define MM_F_AVA         0x002
-#define MM_F_CIGAR       0x004
-#define MM_F_OUT_SAM     0x008
-#define MM_F_NO_QUAL     0x010
-#define MM_F_OUT_CG      0x020
-#define MM_F_OUT_CS      0x040
-#define MM_F_SPLICE      0x080
-#define MM_F_SPLICE_FOR  0x100
-#define MM_F_SPLICE_REV  0x200
-#define MM_F_SPLICE_BOTH 0x400
-#define MM_F_NO_SAM_SQ   0x800
+#define MM_I_HPC          0x1
+#define MM_I_NO_SEQ       0x2
+#define MM_I_NO_NAME      0x4
 
 #define MM_IDX_MAGIC   "MMI\2"
 
+#define MM_MAX_SEG       255
+
 #ifdef __cplusplus
 extern "C" {
 #endif
 
-typedef struct {
- 	uint64_t x, y;
-} mm128_t;
-
+// emulate 128-bit integers and arrays
+typedef struct { uint64_t x, y; } mm128_t;
 typedef struct { size_t n, m; mm128_t *a; } mm128_v;
-typedef struct { size_t n, m; uint64_t *a; } uint64_v;
-typedef struct { size_t n, m; uint32_t *a; } uint32_v;
-
-typedef struct {
-	mm128_v a;   // (minimizer, position) array
-	int32_t n;   // size of the _p_ array
-	uint64_t *p; // position array for minimizers appearing >1 times
-	void *h;     // hash table indexing _p_ and minimizers appearing once
-} mm_idx_bucket_t;
 
+// minimap2 index
 typedef struct {
 	char *name;      // name of the db sequence
 	uint64_t offset; // offset in mm_idx_t::S
@@ -48,104 +60,311 @@ typedef struct {
 } mm_idx_seq_t;
 
 typedef struct {
-	int32_t b, w, k, is_hpc;
-	uint32_t n_seq;     // number of reference sequences
-	mm_idx_seq_t *seq;  // sequence name, length and offset
-	uint32_t *S;        // 4-bit packed sequence
-	mm_idx_bucket_t *B; // index
-	void *km;
+	int32_t b, w, k, flag;
+	uint32_t n_seq;            // number of reference sequences
+	int32_t index;
+	mm_idx_seq_t *seq;         // sequence name, length and offset
+	uint32_t *S;               // 4-bit packed sequence
+	struct mm_idx_bucket_s *B; // index (hidden)
+	void *km, *h;
 } mm_idx_t;
 
+// minimap2 alignment
 typedef struct {
-	uint32_t capacity;
-	int32_t dp_score, dp_max, dp_max2;
-	uint32_t blen;
-	uint32_t n_diff;
-	uint32_t n_ambi:30, trans_strand:2;
-	uint32_t n_cigar;
+	uint32_t capacity;                  // the capacity of cigar[]
+	int32_t dp_score, dp_max, dp_max2;  // DP score; score of the max-scoring segment; score of the best alternate mappings
+	uint32_t n_ambi:30, trans_strand:2; // number of ambiguous bases; transcript strand: 0 for unknown, 1 for +, 2 for -
+	uint32_t n_cigar;                   // number of cigar operations in cigar[]
 	uint32_t cigar[];
 } mm_extra_t;
 
 typedef struct {
-	int32_t id;
-	uint32_t cnt:31, rev:1;
-	uint32_t rid:31, inv:1;
-	int32_t score;
-	int32_t qs, qe, rs, re;
-	int32_t parent, subsc;
-	int32_t as;
-	int32_t fuzzy_mlen, fuzzy_blen;
-	uint32_t mapq:8, split:2, sam_pri:1, n_sub:21; // TODO: n_sub is not used for now
+	int32_t id;             // ID for internal uses (see also parent below)
+	int32_t cnt;            // number of minimizers; if on the reverse strand
+	int32_t rid;            // reference index; if this is an alignment from inversion rescue
+	int32_t score;          // DP alignment score
+	int32_t qs, qe, rs, re; // query start and end; reference start and end
+	int32_t parent, subsc;  // parent==id if primary; best alternate mapping score
+	int32_t as;             // offset in the a[] array (for internal uses only)
+	int32_t mlen, blen;     // seeded exact match length; seeded alignment block length
+	int32_t n_sub;          // number of suboptimal mappings
+	int32_t score0;         // initial chaining score (before chain merging/spliting)
+	uint32_t mapq:8, split:2, rev:1, inv:1, sam_pri:1, proper_frag:1, pe_thru:1, seg_split:1, seg_id:8, split_inv:1, dummy:7;
+	uint32_t hash;
+	float div;
 	mm_extra_t *p;
 } mm_reg1_t;
 
+// indexing and mapping options
 typedef struct {
-	float max_occ_frac;
-	float mid_occ_frac;
-	int sdust_thres;  // score threshold for SDUST; 0 to disable
-	int flag;    // see MM_F_* macros
+	short k, w, flag, bucket_bits;
+	int mini_batch_size;
+	uint64_t batch_size;
+} mm_idxopt_t;
 
-	int bw;  // bandwidth
+typedef struct {
+	int seed;
+	int sdust_thres; // score threshold for SDUST; 0 to disable
+	int flag;        // see MM_F_* macros
+
+	int bw;          // bandwidth
 	int max_gap, max_gap_ref; // break a chain if there are no minimizers in a max_gap window
+	int max_frag_len;
 	int max_chain_skip;
-	int min_cnt;
-	int min_chain_score;
+	int min_cnt;         // min number of minimizers on each chain
+	int min_chain_score; // min chaining score
 
 	float mask_level;
 	float pri_ratio;
-	int best_n;
+	int best_n;      // top best_n chains are subjected to DP alignment
 
 	int max_join_long, max_join_short;
 	int min_join_flank_sc;
+	float min_join_flank_ratio;
 
 	int a, b, q, e, q2, e2; // matching score, mismatch, gap-open and gap-ext penalties
-	int noncan;
-	int zdrop;
-	int min_dp_max;
+	int sc_ambi; // score when one or both bases are "N"
+	int noncan;      // cost of non-canonical splicing sites
+	int zdrop, zdrop_inv;   // break alignment if alignment score drops too fast along the diagonal
+	int end_bonus;
+	int min_dp_max;  // drop an alignment if the score of the max scoring segment is below this threshold
 	int min_ksw_len;
+	int anchor_ext_len, anchor_ext_shift;
+	float max_clip_ratio; // drop an alignment if BOTH ends are clipped above this ratio
 
-	int max_occ;
-	int mid_occ;
+	int pe_ori, pe_bonus;
+
+	float mid_occ_frac;  // only used by mm_mapopt_update(); see below
+	int32_t min_mid_occ;
+	int32_t mid_occ;     // ignore seeds with occurrences above this threshold
+	int32_t max_occ;
+	int mini_batch_size; // size of a batch of query bases to process in parallel
+
+	const char *split_prefix;
 } mm_mapopt_t;
 
-extern int mm_verbose, mm_dbg_flag;
-extern double mm_realtime0;
+// index reader
+typedef struct {
+	int is_idx, n_parts;
+	int64_t idx_size;
+	mm_idxopt_t opt;
+	FILE *fp_out;
+	union {
+		struct mm_bseq_file_s *seq;
+		FILE *idx;
+	} fp;
+} mm_idx_reader_t;
 
-struct mm_tbuf_s;
+// memory buffer for thread-local storage during mapping
 typedef struct mm_tbuf_s mm_tbuf_t;
 
-struct mm_bseq_file_s;
+// global variables
+extern int mm_verbose, mm_dbg_flag; // verbose level: 0 for no info, 1 for error, 2 for warning, 3 for message (default); debugging flag
+extern double mm_realtime0; // wall-clock timer
 
-#define mm_seq4_set(s, i, c) ((s)[(i)>>3] |= (uint32_t)(c) << (((i)&7)<<2))
-#define mm_seq4_get(s, i)    ((s)[(i)>>3] >> (((i)&7)<<2) & 0xf)
+/**
+ * Set default or preset parameters
+ *
+ * @param preset     NULL to set all parameters as default; otherwise apply preset to affected parameters
+ * @param io         pointer to indexing parameters
+ * @param mo         pointer to mapping parameters
+ *
+ * @return 0 if success; -1 if _present_ unknown
+ */
+int mm_set_opt(const char *preset, mm_idxopt_t *io, mm_mapopt_t *mo);
+int mm_check_opt(const mm_idxopt_t *io, const mm_mapopt_t *mo);
 
-// compute minimizers
-void mm_sketch(void *km, const char *str, int len, int w, int k, uint32_t rid, int is_hpc, mm128_v *p);
+/**
+ * Update mm_mapopt_t::mid_occ via mm_mapopt_t::mid_occ_frac
+ *
+ * If mm_mapopt_t::mid_occ is 0, this function sets it to a number such that no
+ * more than mm_mapopt_t::mid_occ_frac of minimizers in the index have a higher
+ * occurrence.
+ *
+ * @param opt        mapping parameters
+ * @param mi         minimap2 index
+ */
+void mm_mapopt_update(mm_mapopt_t *opt, const mm_idx_t *mi);
 
-// minimizer indexing
-mm_idx_t *mm_idx_init(int w, int k, int b, int is_hpc);
-void mm_idx_destroy(mm_idx_t *mi);
-mm_idx_t *mm_idx_gen(struct mm_bseq_file_s *fp, int w, int k, int b, int is_hpc, int mini_batch_size, int n_threads, uint64_t batch_size, int keep_name);
-uint32_t mm_idx_cal_max_occ(const mm_idx_t *mi, float f);
-void mm_idx_stat(const mm_idx_t *idx);
-const uint64_t *mm_idx_get(const mm_idx_t *mi, uint64_t minier, int *n);
-int mm_idx_getseq(const mm_idx_t *mi, uint32_t rid, uint32_t st, uint32_t en, uint8_t *seq);
+void mm_mapopt_max_intron_len(mm_mapopt_t *opt, int max_intron_len);
 
-mm_idx_t *mm_idx_build(const char *fn, int w, int k, int is_hpc, int n_threads);
-int mm_idx_is_idx(const char *fn);
+/**
+ * Initialize an index reader
+ *
+ * @param fn         index or fasta/fastq file name (this function tests the file type)
+ * @param opt        indexing parameters
+ * @param fn_out     if not NULL, write built index to this file
+ *
+ * @return an index reader on success; NULL if fail to open _fn_
+ */
+mm_idx_reader_t *mm_idx_reader_open(const char *fn, const mm_idxopt_t *opt, const char *fn_out);
 
-// minimizer index I/O
-void mm_idx_dump(FILE *fp, const mm_idx_t *mi);
+/**
+ * Read/build an index
+ *
+ * If the input file is an index file, this function reads one part of the
+ * index and returns. If the input file is a sequence file (fasta or fastq),
+ * this function constructs the index for about mm_idxopt_t::batch_size bases.
+ * Importantly, for a huge collection of sequences, this function may only
+ * return an index for part of sequences. It needs to be repeatedly called
+ * to traverse the entire index/sequence file.
+ *
+ * @param r          index reader
+ * @param n_threads  number of threads for constructing index
+ *
+ * @return an index on success; NULL if reaching the end of the input file
+ */
+mm_idx_t *mm_idx_reader_read(mm_idx_reader_t *r, int n_threads);
+
+/**
+ * Destroy/deallocate an index reader
+ *
+ * @param r          index reader
+ */
+void mm_idx_reader_close(mm_idx_reader_t *r);
+
+int mm_idx_reader_eof(const mm_idx_reader_t *r);
+
+/**
+ * Check whether the file contains a minimap2 index
+ *
+ * @param fn         file name
+ *
+ * @return the file size if fn is an index file; 0 if fn is not.
+ */
+int64_t mm_idx_is_idx(const char *fn);
+
+/**
+ * Load a part of an index
+ *
+ * Given a uni-part index, this function loads the entire index into memory.
+ * Given a multi-part index, it loads one part only and places the file pointer
+ * at the end of that part.
+ *
+ * @param fp         pointer to FILE object
+ *
+ * @return minimap2 index read from fp
+ */
 mm_idx_t *mm_idx_load(FILE *fp);
 
-// mapping
-void mm_mapopt_init(mm_mapopt_t *opt);
-void mm_mapopt_update(mm_mapopt_t *opt, const mm_idx_t *mi);
+/**
+ * Append an index (or one part of a full index) to file
+ *
+ * @param fp         pointer to FILE object
+ * @param mi         minimap2 index
+ */
+void mm_idx_dump(FILE *fp, const mm_idx_t *mi);
+
+/**
+ * Create an index from strings in memory
+ *
+ * @param w            minimizer window size
+ * @param k            minimizer k-mer size
+ * @param is_hpc       use HPC k-mer if true
+ * @param bucket_bits  number of bits for the first level of the hash table
+ * @param n            number of sequences
+ * @param seq          sequences in A/C/G/T
+ * @param name         sequence names; could be NULL
+ *
+ * @return minimap2 index
+ */
+mm_idx_t *mm_idx_str(int w, int k, int is_hpc, int bucket_bits, int n, const char **seq, const char **name);
+
+/**
+ * Print index statistics to stderr
+ *
+ * @param mi         minimap2 index
+ */
+void mm_idx_stat(const mm_idx_t *idx);
+
+/**
+ * Destroy/deallocate an index
+ *
+ * @param r          minimap2 index
+ */
+void mm_idx_destroy(mm_idx_t *mi);
+
+/**
+ * Initialize a thread-local buffer for mapping
+ *
+ * Each mapping thread requires a buffer specific to the thread (see mm_map()
+ * below). The primary purpose of this buffer is to reduce frequent heap
+ * allocations across threads. A buffer shall not be used by two or more
+ * threads.
+ *
+ * @return pointer to a thread-local buffer
+ */
 mm_tbuf_t *mm_tbuf_init(void);
+
+/**
+ * Destroy/deallocate a thread-local buffer for mapping
+ *
+ * @param b          the buffer
+ */
 void mm_tbuf_destroy(mm_tbuf_t *b);
+
+void *mm_tbuf_get_km(mm_tbuf_t *b);
+
+/**
+ * Align a query sequence against an index
+ *
+ * This function possibly finds multiple alignments of the query sequence.
+ * The returned array and the mm_reg1_t::p field of each element are allocated
+ * with malloc().
+ *
+ * @param mi         minimap2 index
+ * @param l_seq      length of the query sequence
+ * @param seq        the query sequence
+ * @param n_regs     number of hits (out)
+ * @param b          thread-local buffer; two mm_map() calls shall not use one buffer at the same time!
+ * @param opt        mapping parameters
+ * @param name       query name, used for all-vs-all overlapping and debugging
+ *
+ * @return an array of hits which need to be deallocated with free() together
+ *         with mm_reg1_t::p of each element. The size is written to _n_regs_.
+ */
 mm_reg1_t *mm_map(const mm_idx_t *mi, int l_seq, const char *seq, int *n_regs, mm_tbuf_t *b, const mm_mapopt_t *opt, const char *name);
 
-int mm_map_file(const mm_idx_t *idx, const char *fn, const mm_mapopt_t *opt, int n_threads, int tbatch_size);
+void mm_map_frag(const mm_idx_t *mi, int n_segs, const int *qlens, const char **seqs, int *n_regs, mm_reg1_t **regs, mm_tbuf_t *b, const mm_mapopt_t *opt, const char *qname);
+
+/**
+ * Align a fasta/fastq file and print alignments to stdout
+ *
+ * @param idx        minimap2 index
+ * @param fn         fasta/fastq file name
+ * @param opt        mapping parameters
+ * @param n_threads  number of threads
+ *
+ * @return 0 on success; -1 if _fn_ can't be read
+ */
+int mm_map_file(const mm_idx_t *idx, const char *fn, const mm_mapopt_t *opt, int n_threads);
+
+int mm_map_file_frag(const mm_idx_t *idx, int n_segs, const char **fn, const mm_mapopt_t *opt, int n_threads);
+
+/**
+ * Generate the cs tag (new in 2.12)
+ *
+ * @param km         memory blocks; set to NULL if unsure
+ * @param buf        buffer to write the cs/MD tag; typicall NULL on the first call
+ * @param max_len    max length of the buffer; typically set to 0 on the first call
+ * @param mi         index
+ * @param r          alignment
+ * @param seq        query sequence
+ * @param no_iden    true to use : instead of =
+ *
+ * @return the length of cs
+ */
+int mm_gen_cs(void *km, char **buf, int *max_len, const mm_idx_t *mi, const mm_reg1_t *r, const char *seq, int no_iden);
+int mm_gen_MD(void *km, char **buf, int *max_len, const mm_idx_t *mi, const mm_reg1_t *r, const char *seq);
+
+// query sequence name and sequence in the minimap2 index
+int mm_idx_index_name(mm_idx_t *mi);
+int mm_idx_name2id(const mm_idx_t *mi, const char *name);
+int mm_idx_getseq(const mm_idx_t *mi, uint32_t rid, uint32_t st, uint32_t en, uint8_t *seq);
+
+// deprecated APIs for backward compatibility
+void mm_mapopt_init(mm_mapopt_t *opt);
+mm_idx_t *mm_idx_build(const char *fn, int w, int k, int flag, int n_threads);
 
 #ifdef __cplusplus
 }

minimap2.1

@@ -1,4 +1,4 @@
-.TH minimap2 1 "25 August 2017" "minimap2-2.1-r311" "Bioinformatics tools"
+.TH minimap2 1 "11 October 2018" "minimap2-2.13 (r850)" "Bioinformatics tools"
 .SH NAME
 .PP
 minimap2 - mapping and alignment between collections of DNA sequences
@@ -99,6 +99,14 @@ multiple times to map it against each batch of target sequences.
 may be ending with k/K/m/M/g/G. NB: mapping quality is incorrect given a
 multi-part index.
 .TP
+.B --idx-no-seq
+Don't store target sequences in the index. It saves disk space and memory but
+the index generated with this option will not work with
+.B -a
+or
+.BR -c .
+When base-level alignment is not requested, this option is automatically applied.
+.TP
 .BI -d \ FILE
 Save the minimizer index of
 .I target.fa
@@ -115,18 +123,35 @@ provided as the target sequences, options
 will be effectively overridden by the options stored in the index file.
 .SS Mapping options
 .TP 10
-.BI -f \ FLOAT
-Ignore top
+.BI -f \ FLOAT | INT1 [, INT2 ]
+If fraction, ignore top
 .I FLOAT
-fraction of most frequent minimizers [0.0002]
+fraction of most frequent minimizers [0.0002]. If integer,
+ignore minimizers occuring more than
+.I INT1
+times.
+.I INT2
+is only effective in the
+.B --sr
+or
+.B -xsr
+mode, which sets the threshold for a second round of seeding.
+.TP
+.BI --min-occ-floor \ INT
+Force minimap2 to always use k-mers occurring
+.I INT
+times or less [0]. In effect, the max occurrence threshold is set to
+the
+.RI max{ INT ,
+.BR -f }.
 .TP
 .BI -g \ INT
-Stop chain enlongation if there are no minimizers in
+Stop chain enlongation if there are no minimizers within
 .IR INT -bp
 [10000].
 .TP
 .BI -r \ INT
-Bandwidth used in chaining and DP-based alignment [1000]. This option
+Bandwidth used in chaining and DP-based alignment [500]. This option
 approximately controls the maximum gap size.
 .TP
 .BI -n \ INT
@@ -140,20 +165,42 @@ Discard chains with chaining score
 [40]. Chaining score equals the approximate number of matching bases minus a
 concave gap penalty. It is computed with dynamic programming.
 .TP
+.B -D
+If query sequence name/length are identical to the target name/length, ignore
+diagonal anchors. This option also reduces DP-based extension along the
+diagonal.
+.TP
+.B -P
+Retain all chains and don't attempt to set primary chains. Options
+.B -p
+and
+.B -N
+have no effect when this option is in use.
+.TP
+.BR --dual = yes | no
+If
+.BR no ,
+skip query-target pairs wherein the query name is lexicographically greater
+than the target name [yes]
+.TP
 .B -X
-Perform all-vs-all mapping. In this mode, if the query sequence name is
-lexicographically larger than the target sequence name, the hits between them
-will be suppressed; if the query sequence name is the same as the target name,
-diagonal minimizer hits will also be suppressed.
+Equivalent to
+.RB ' -DP
+.BR --dual = no
+.BR --no-long-join '.
+Primarily used for all-vs-all read overlapping.
 .TP
 .BI -p \ FLOAT
 Minimal secondary-to-primary score ratio to output secondary mappings [0.8].
-Between two chains overlaping over half of the shorter chain (controled by
-.BR --mask-level ),
+Between two chains overlaping over half of the shorter chain (controlled by
+.BR -M ),
 the chain with a lower score is secondary to the chain with a higher score.
 If the ratio of the scores is below
 .IR FLOAT ,
 the secondary chain will not be outputted or extended with DP alignment later.
+This option has no effect when
+.B -X
+is applied.
 .TP
 .BI -N \ INT
 Output at most
@@ -163,10 +210,26 @@ secondary alignments [5]. This option has no effect when
 is applied.
 .TP
 .BI -G \ NUM
-Maximal intron length in the splice mode [200k]. This option also changes the
-bandwidth to
+Maximum gap on the reference (effective with
+.BR -xsplice / --splice ).
+This option also changes the chaining and alignment band width to
 .IR NUM .
-Increasing this option slows down spliced alignment.
+Increasing this option slows down spliced alignment. [200k]
+.TP
+.BI -F \ NUM
+Maximum fragment length (aka insert size; effective with
+.BR -xsr / --frag = yes )
+[800]
+.TP
+.BI -M \ FLOAT
+Mark as secondary a chain that overlaps with a better chain by
+.I FLOAT
+or more of the shorter chain [0.5]
+.TP
+.B --hard-mask-level
+Honor option
+.B -M
+and disable a heurstic to save unmapped subsequences.
 .TP
 .BI --max-chain-skip \ INT
 A heuristics that stops chaining early [50]. Minimap2 uses dynamic programming
@@ -175,6 +238,42 @@ option makes minimap2 exits the inner loop if it repeatedly sees seeds already
 on chains. Set
 .I INT
 to a large number to switch off this heurstics.
+.TP
+.B --no-long-join
+Disable the long gap patching heuristic. When this option is applied, the
+maximum alignment gap is mostly controlled by
+.BR -r .
+.TP
+.BI --lj-min-ratio \ FLOAT
+Fraction of query sequence length required to bridge a long gap [0.5]. A
+smaller value helps to recover longer gaps, at the cost of more false gaps.
+.TP
+.B --splice
+Enable the splice alignment mode.
+.TP
+.B --sr
+Enable short-read alignment heuristics. In the short-read mode, minimap2
+applies a second round of chaining with a higher minimizer occurrence threshold
+if no good chain is found. In addition, minimap2 attempts to patch gaps between
+seeds with ungapped alignment.
+.TP
+.BI --split-prefix \ STR
+Prefix to create temporary files. Typically used for a multi-part index.
+.TP
+.BR --frag = no | yes
+Whether to enable the fragment mode [no]
+.TP
+.B --for-only
+Only map to the forward strand of the reference sequences. For paired-end
+reads in the forward-reverse orientation, the first read is mapped to forward
+strand of the reference and the second read to the reverse stand.
+.TP
+.B --rev-only
+Only map to the reverse complement strand of the reference sequences.
+.TP
+.BR --heap-sort = no | yes
+If yes, sort anchors with heap merge, instead of radix sort. Heap merge is
+faster for short reads, but slower for long reads. [no]
 .SS Alignment options
 .TP 10
 .BI -A \ INT
@@ -194,12 +293,29 @@ Gap extension penalty [2,1]. A gap of length
 .I k
 costs
 .RI min{ O1 + k * E1 , O2 + k * E2 }.
+In the splice mode, the second gap penalties are not used.
 .TP
-.BI -z \ INT
-Break an alignment if the running score drops too quickly along the diagonal of
-the DP matrix (diagonal X-drop, or Z-drop) [400]. Increasing the value improves
-the contiguity of the alignment at the cost of poor alignment in the middle
-(e.g. caused by a long inversion).
+.BI -C \ INT
+Cost for a non-canonical GT-AG splicing (effective with
+.BR --splice )
+[0]
+.TP
+.BI -z \ INT1[,INT2]
+Truncate an alignment if the running alignment score drops too quickly along
+the diagonal of the DP matrix (diagonal X-drop, or Z-drop) [400,200]. If the
+drop of score is above
+.IR INT2 ,
+minimap2 will reverse complement the query in the related region and align
+again to test small inversions. Minimap2 truncates alignment if there is an
+inversion or the drop of score is greater than
+.IR INT1 .
+Decrease
+.I INT2
+to find small inversions at the cost of performance and false positives.
+Increase
+.I INT1
+to improves the contiguity of alignment at the cost of poor alignment in the
+middle.
 .TP
 .BI -s \ INT
 Minimal peak DP alignment score to output [40]. The peak score is computed from
@@ -215,8 +331,44 @@ both strands;
 .BR n :
 no attempt to match GT-AG [n]
 .TP
-.BI --cost-non-gt-ag \ INT
-Cost of non-canonical splicing sites [0].
+.BI --end-bonus \ INT
+Score bonus when alignment extends to the end of the query sequence [0].
+.TP
+.BI --score-N \ INT
+Score of a mismatch involving ambiguous bases [1].
+.TP
+.BR --splice-flank = yes | no
+Assume the next base to a
+.B GT
+donor site tends to be A/G (91% in human and 92% in mouse) and the preceding
+base to a
+.B AG
+acceptor tends to be C/T [no].
+This trend is evolutionarily conservative, all the way to S. cerevisiae
+(PMID:18688272). Specifying this option generally leads to higher junction
+accuracy by several percents, so it is applied by default with
+.BR --splice .
+However, the SIRV control does not honor this trend
+(only ~60%). This option reduces accuracy. If you are benchmarking minimap2
+on SIRV data, please add
+.B --splice-flank=no
+to the command line.
+.TP
+.BI --end-seed-pen \ INT
+Drop a terminal anchor if
+.IR s <log( g )+ INT ,
+where
+.I s
+is the local alignment score around the anchor and
+.I g
+the length of the terminal gap in the chain. This option is only effective
+with
+.BR --splice .
+It helps to avoid tiny terminal exons. [6]
+.TP
+.B --no-end-flt
+Don't filter seeds towards the ends of chains before performing base-level
+alignment.
 .SS Input/output options
 .TP 10
 .B -a
@@ -226,14 +378,52 @@ by default.
 .B -Q
 Ignore base quality in the input file.
 .TP
+.B -L
+Write CIGAR with >65535 operators at the CG tag. Older tools are unable to
+convert alignments with >65535 CIGAR ops to BAM. This option makes minimap2 SAM
+compatible with older tools. Newer tools recognizes this tag and reconstruct
+the real CIGAR in memory.
+.TP
 .BI -R \ STR
 SAM read group line in a format like
 .B @RG\\\\tID:foo\\\\tSM:bar
 [].
 .TP
+.B -y
+Copy input FASTA/Q comments to output.
+.TP
 .B -c
 Generate CIGAR. In PAF, the CIGAR is written to the `cg' custom tag.
 .TP
+.BI --cs[= STR ]
+Output the
+.B cs
+tag.
+.I STR
+can be either
+.I short
+or
+.IR long .
+If no
+.I STR
+is given,
+.I short
+is assumed. [none]
+.TP
+.B --MD
+Output the MD tag (see the SAM spec).
+.TP
+.B --eqx
+Output =/X CIGAR operators for sequence match/mismatch.
+.TP
+.B -Y
+In SAM output, use soft clipping for supplementary alignments.
+.TP
+.BI --seed \ INT
+Integer seed for randomizing equally best hits. Minimap2 hashes
+.I INT
+and read name when choosing between equally best hits. [11]
+.TP
 .BI -t \ INT
 Number of threads [3]. Minimap2 uses at most three threads when indexing target
 sequences, and uses up to
@@ -241,27 +431,26 @@ sequences, and uses up to
 threads when mapping (the extra thread is for I/O, which is frequently idle and
 takes little CPU time).
 .TP
+.B -2
+Use two I/O threads during mapping. By default, minimap2 uses one I/O thread.
+When I/O is slow (e.g. piping to gzip, or reading from a slow pipe), the I/O
+thread may become the bottleneck. Apply this option to use one thread for input
+and another thread for output, at the cost of increased peak RAM.
+.TP
 .BI -K \ NUM
-Number of bases loaded into memory to process in a mini-batch [200M].
+Number of bases loaded into memory to process in a mini-batch [500M].
 Similar to option
 .BR -I ,
 K/M/G/k/m/g suffix is accepted. A large
 .I NUM
 helps load balancing in the multi-threading mode, at the cost of increased
-memory. Preset
-.B ava-pb
-and
-.B ava-ont
-use
-.BR -K500m .
+memory.
+.TP
+.BR --secondary = yes | no
+Whether to output secondary alignments [yes]
 .TP
 .B --version
 Print version number to stdout
-.TP
-.B --no-sam-hdr
-Don't output SAM header lines. Use this option if the index consists of
-multiple parts; otherwise the SAM output is malformated due to internal header
-lines.
 .SS Preset options
 .TP 10
 .BI -x \ STR
@@ -278,11 +467,6 @@ are:
 PacBio/Oxford Nanopore read to reference mapping
 .RB ( -Hk19 )
 .TP
-.B map10k
-The same as
-.B map-pb
-.RB ( -Hk19 )
-.TP
 .B map-ont
 Slightly more sensitive for Oxford Nanopore to reference mapping
 .RB ( -k15 ).
@@ -294,28 +478,35 @@ is determined by the sequencing error mode.
 .B asm5
 Long assembly to reference mapping
 .RB ( -k19
-.B -w19 -A1 -B19 -O39,81 -E3,1 -s200
-.BR -z200 ).
+.B -w19 -A1 -B19 -O39,81 -E3,1 -s200 -z200
+.BR --min-occ-floor=100 ).
 Typically, the alignment will not extend to regions with 5% or higher sequence
 divergence. Only use this preset if the average divergence is far below 5%.
 .TP
 .B asm10
 Long assembly to reference mapping
 .RB ( -k19
-.B -w19 -A1 -B9 -O16,41 -E2,1 -s200
-.BR -z200 ).
+.B -w19 -A1 -B9 -O16,41 -E2,1 -s200 -z200
+.BR --min-occ-floor=100 ).
 Up to 10% sequence divergence.
 .TP
+.B asm20
+Long assembly to reference mapping
+.RB ( -k19
+.B -w10 -A1 -B6 -O6,26 -E2,1 -s200 -z200
+.BR --min-occ-floor=100 ).
+Up to 20% sequence divergence.
+.TP
 .B ava-pb
 PacBio all-vs-all overlap mapping
 .RB ( -Hk19
-.B -w5 -Xp0 -m100 -K500m -g10000 --max-chain-skip
+.B -Xw5 -m100 -g10000 --max-chain-skip
 .BR 25 ).
 .TP
 .B ava-ont
 Oxford Nanopore all-vs-all overlap mapping
 .RB ( -k15
-.B -w5 -Xp0 -m100 -K500m -g10000 --max-chain-skip
+.B -Xw5 -m100 -g10000 -r2000 --max-chain-skip
 .BR 25 ).
 Similarly, the major difference from
 .B ava-pb
@@ -324,8 +515,8 @@ is that this preset is not using HPC minimizers.
 .B splice
 Long-read spliced alignment
 .RB ( -k15
-.B -w5 --splice -g2000 -G200k -A1 -B2 -O2,32 -E1,0 -z200 -ub --cost-non-gt-ag
-.BR 5 ).
+.B -w5 --splice -g2000 -G200k -A1 -B2 -O2,32 -E1,0 -C9 -z200 -ub
+.BR --splice-flank=yes ).
 In the splice mode, 1) long deletions are taken as introns and represented as
 the
 .RB ` N '
@@ -333,6 +524,13 @@ CIGAR operator; 2) long insertions are disabled; 3) deletion and insertion gap
 costs are different during chaining; 4) the computation of the
 .RB ` ms '
 tag ignores introns to demote hits to pseudogenes.
+.TP
+.B sr
+Short single-end reads without splicing
+.RB ( -k21
+.B -w11 --sr --frag=yes -A2 -B8 -O12,32 -E2,1 -r50 -p.5 -N20 -f1000,5000 -n2 -m20
+.B -s40 -g200 -2K50m --heap-sort=yes
+.BR --secondary=no ).
 .RE
 .SS Miscellaneous options
 .TP 10
@@ -345,7 +543,7 @@ multi-threading mode.
 .B --print-qname
 Print query names to stderr, mostly to see which query is crashing minimap2.
 .TP
-.B --print-seed
+.B --print-seeds
 Print seed positions to stderr, for debugging only.
 .SH OUTPUT FORMAT
 .PP
@@ -384,15 +582,39 @@ cb | cb | cb
 r | c | l .
 Tag	Type	Description
 _
-tp	A	Type of aln: P/primary, S/secondary and I/inversion
+tp	A	Type of aln: P/primary, S/secondary and I,i/inversion
 cm	i	Number of minimizers on the chain
 s1	i	Chaining score
 s2	i	Chaining score of the best secondary chain
 NM	i	Total number of mismatches and gaps in the alignment
+MD	Z	To generate the ref sequence in the alignment
 AS	i	DP alignment score
 ms	i	DP score of the max scoring segment in the alignment
 nn	i	Number of ambiguous bases in the alignment
+ts	A	Transcript strand (splice mode only)
 cg	Z	CIGAR string (only in PAF)
+cs	Z	Difference string
+dv	f	Approximate per-base sequence divergence
+.TE
+
+.PP
+The
+.B cs
+tag encodes difference sequences in the short form or the entire query
+.I AND
+reference sequences in the long form. It consists of a series of operations:
+.TS
+center box;
+cb | cb |cb
+r | l | l .
+Op	Regex	Description
+_
+ =	[ACGTN]+	Identical sequence (long form)
+ :	[0-9]+	Identical sequence length
+ *	[acgtn][acgtn]	Substitution: ref to query
+ +	[acgtn]+	Insertion to the reference
+ -	[acgtn]+	Deletion from the reference
+ ~	[acgtn]{2}[0-9]+[acgtn]{2}	Intron length and splice signal
 .TE
 
 .SH LIMITATIONS
@@ -403,11 +625,8 @@ where seed positions may be suboptimal. This should not be a big concern
 because even the optimal alignment may be wrong in such regions.
 .TP
 *
-Minimap2 does not work well with Illumina short reads as of now.
-.TP
-*
-Minimap2 requires SSE2 instructions to compile. It is possible to add
-non-SSE2 support, but it would make minimap2 slower by several times.
+Minimap2 requires SSE2 or NEON instructions to compile. It is possible to add
+non-SSE2/NEON support, but it would make minimap2 slower by several times.
 .SH SEE ALSO
 .PP
 miniasm(1), minimap(1), bwa(1).

misc.c

@@ -1,19 +1,119 @@
-#include <sys/resource.h>
-#include <sys/time.h>
-#include "minimap.h"
+#include <stdlib.h>
+#include "mmpriv.h"
 
-int mm_verbose = 3;
+int mm_verbose = 1;
 int mm_dbg_flag = 0;
 double mm_realtime0;
 
+#if defined(WIN32) || defined(_WIN32)
+#include <windows.h>
+
+struct timezone
+{
+  __int32  tz_minuteswest; /* minutes W of Greenwich */
+  int  tz_dsttime;     /* type of dst correction */
+};
+
+/*
+ * gettimeofday.c
+ *    Win32 gettimeofday() replacement
+ *    taken from PostgreSQL, according to
+ *    https://stackoverflow.com/questions/1676036/what-should-i-use-to-replace-gettimeofday-on-windows
+ *
+ * src/port/gettimeofday.c
+ *
+ * Copyright (c) 2003 SRA, Inc.
+ * Copyright (c) 2003 SKC, Inc.
+ *
+ * Permission to use, copy, modify, and distribute this software and
+ * its documentation for any purpose, without fee, and without a
+ * written agreement is hereby granted, provided that the above
+ * copyright notice and this paragraph and the following two
+ * paragraphs appear in all copies.
+ *
+ * IN NO EVENT SHALL THE AUTHOR BE LIABLE TO ANY PARTY FOR DIRECT,
+ * INDIRECT, SPECIAL, INCIDENTAL, OR CONSEQUENTIAL DAMAGES, INCLUDING
+ * LOST PROFITS, ARISING OUT OF THE USE OF THIS SOFTWARE AND ITS
+ * DOCUMENTATION, EVEN IF THE UNIVERSITY OF CALIFORNIA HAS BEEN ADVISED
+ * OF THE POSSIBILITY OF SUCH DAMAGE.
+ *
+ * THE AUTHOR SPECIFICALLY DISCLAIMS ANY WARRANTIES, INCLUDING, BUT NOT
+ * LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR
+ * A PARTICULAR PURPOSE.  THE SOFTWARE PROVIDED HEREUNDER IS ON AN "AS
+ * IS" BASIS, AND THE AUTHOR HAS NO OBLIGATIONS TO PROVIDE MAINTENANCE,
+ * SUPPORT, UPDATES, ENHANCEMENTS, OR MODIFICATIONS.
+ */
+
+/* FILETIME of Jan 1 1970 00:00:00. */
+static const unsigned __int64 epoch = ((unsigned __int64) 116444736000000000ULL);
+
+/*
+ * timezone information is stored outside the kernel so tzp isn't used anymore.
+ *
+ * Note: this function is not for Win32 high precision timing purpose. See
+ * elapsed_time().
+ */
+int gettimeofday(struct timeval * tp, struct timezone *tzp)
+{
+    FILETIME    file_time;
+    SYSTEMTIME  system_time;
+    ULARGE_INTEGER ularge;
+
+    GetSystemTime(&system_time);
+    SystemTimeToFileTime(&system_time, &file_time);
+    ularge.LowPart = file_time.dwLowDateTime;
+    ularge.HighPart = file_time.dwHighDateTime;
+
+    tp->tv_sec = (long) ((ularge.QuadPart - epoch) / 10000000L);
+    tp->tv_usec = (long) (system_time.wMilliseconds * 1000);
+
+    return 0;
+}
+
+// taken from https://stackoverflow.com/questions/5272470/c-get-cpu-usage-on-linux-and-windows
 double cputime()
+{
+	HANDLE hProcess = GetCurrentProcess();
+	FILETIME ftCreation, ftExit, ftKernel, ftUser;
+	SYSTEMTIME stKernel;
+	SYSTEMTIME stUser;
+
+	GetProcessTimes(hProcess, &ftCreation, &ftExit, &ftKernel, &ftUser);
+	FileTimeToSystemTime(&ftKernel, &stKernel);
+	FileTimeToSystemTime(&ftUser, &stUser);
+
+	double kernelModeTime = ((stKernel.wHour * 60.) + stKernel.wMinute * 60.) + stKernel.wSecond * 1. + stKernel.wMilliseconds / 1000.;
+	double userModeTime = ((stUser.wHour * 60.) + stUser.wMinute * 60.) + stUser.wSecond * 1. + stUser.wMilliseconds / 1000.;
+
+	return kernelModeTime + userModeTime;
+}
+
+long peakrss(void) { return 0; }
+#else
+#include <sys/resource.h>
+#include <sys/time.h>
+
+double cputime(void)
 {
 	struct rusage r;
 	getrusage(RUSAGE_SELF, &r);
 	return r.ru_utime.tv_sec + r.ru_stime.tv_sec + 1e-6 * (r.ru_utime.tv_usec + r.ru_stime.tv_usec);
 }
 
-double realtime()
+long peakrss(void)
+{
+	struct rusage r;
+	getrusage(RUSAGE_SELF, &r);
+#ifdef __linux__
+	return r.ru_maxrss * 1024;
+#else
+	return r.ru_maxrss;
+#endif
+}
+
+#endif /* WIN32 || _WIN32 */
+
+double realtime(void)
 {
 	struct timeval tp;
 	struct timezone tzp;
@@ -21,6 +121,36 @@ double realtime()
 	return tp.tv_sec + tp.tv_usec * 1e-6;
 }
 
+void mm_err_puts(const char *str)
+{
+	int ret;
+	ret = puts(str);
+	if (ret == EOF) {
+		fprintf(stderr, "[ERROR] failed to write the results\n");
+		exit(EXIT_FAILURE);
+	}
+}
+
+void mm_err_fwrite(const void *p, size_t size, size_t nitems, FILE *fp)
+{
+	int ret;
+	ret = fwrite(p, size, nitems, fp);
+	if (ret == EOF) {
+		fprintf(stderr, "[ERROR] failed to write data\n");
+		exit(EXIT_FAILURE);
+	}
+}
+
+void mm_err_fread(void *p, size_t size, size_t nitems, FILE *fp)
+{
+	int ret;
+	ret = fread(p, size, nitems, fp);
+	if (ret == EOF) {
+		fprintf(stderr, "[ERROR] failed to read data\n");
+		exit(EXIT_FAILURE);
+	}
+}
+
 #include "ksort.h"
 
 #define sort_key_128x(a) ((a).x)

misc/README.md

@@ -1,28 +1,179 @@
-The [K8 Javascript shell][k8] is needed to run Javascripts in this directory.
-Precompiled k8 binaries for Mac and Linux can be found at the [K8 release
-page][k8bin].
+## <a name="started"></a>Getting Started
 
-* [paf2aln.js](paf2aln.js): convert PAF to [MAF][maf] or BLAST-like output for
-  eyeballing. PAF has to be generated with minimap2 option `-S`, which writes
-  the aligned sequences to the `cs` tag. An example:
-  ```sh
-  ../minimap2 -S ../test/MT-*.fa | k8 paf2aln.js /dev/stdin
-  ```
+```sh
+# install minimap2
+git clone https://github.com/lh3/minimap2
+cd minimap2 && make
+# install the k8 javascript shell
+curl -L https://github.com/attractivechaos/k8/releases/download/v0.2.4/k8-0.2.4.tar.bz2 | tar -jxf -
+cp k8-0.2.4/k8-`uname -s` k8              # or copy it to a directory on your $PATH
+# export PATH="$PATH:`pwd`:`pwd`/misc"    # run this if k8, minimap2 or paftools.js not on your $PATH
+minimap2 --cs test/MT-human.fa test/MT-orang.fa | paftools.js view -     # view alignment
+minimap2 -c test/MT-human.fa test/MT-orang.fa | paftools.js stat -       # basic alignment statistics
+minimap2 -c --cs test/MT-human.fa test/MT-orang.fa \
+  | sort -k6,6 -k8,8n | paftools.js call -L15000 -     # calling variants from asm-to-ref alignment
+minimap2 -c test/MT-human.fa test/MT-orang.fa \
+  | paftools.js liftover -l10000 - <(echo -e "MT_orang\t2000\t5000")     # liftOver
+# no test data for the following examples
+paftools.js junceval -e anno.gtf splice.sam > out.txt  # compare splice junctions to annotations
+paftools.js splice2bed anno.gtf > anno.bed             # convert GTF/GFF3 to BED12
+```
 
-* [mapstat.js](mapstat.js): output basic statistics such as the number of
-  non-redundant mapped bases, number of split and secondary alignments and
-  number of long gaps. This scripts seamlessly works with both SAM and PAF.
+## Table of Contents
 
-* [sim-pbsim.js](sim-pbsim.js): convert reads simulated with [PBSIM][pbsim] to
-  FASTA and encode the true mapping positions to read names in a format like
-  `S1_33!chr1!225258409!225267761!-`.
+- [Getting Started](#started)
+- [Introduction](#intro)
+- [Evaluation](#eval)
+  - [Evaluating mapping accuracy with simulated reads](#mapeval)
+  - [Evaluating read overlap sensitivity](#oveval)
+- [Calling Variants from Assemblies](#asmvar)
 
-* [sim-eval.js](sim-eval.js): evaluate mapping accuracy for FASTA generated
-  with [sim-pbsim.js](sim-pbsim.js) or [sim-mason2.js](sim-mason2.js).
+## <a name="intro"></a>Introduction
 
-* [sam2paf.js](sam2paf.js): convert SAM to PAF.
+paftools.js is a script that processes alignments in the [PAF format][paf],
+such as converting between formats, evaluating mapping accuracy, lifting over
+BED files based on alignment, and calling variants from assembly-to-assembly
+alignment. This script *requires* the [k8 Javascript shell][k8] to run. On
+Linux or Mac, you can download the precompiled k8 binary with:
 
+```sh
+curl -L https://github.com/attractivechaos/k8/releases/download/v0.2.4/k8-0.2.4.tar.bz2 | tar -jxf -
+cp k8-0.2.4/k8-`uname -s` $HOME/bin/k8  # assuming $HOME/bin in your $PATH
+```
+
+It is highly recommended to copy the executable `k8` to a directory on your
+`$PATH` such as `/usr/bin/env` can find it. Like python scripts, once you
+install `k8`, you can launch paftools.js in one of the two ways:
+
+```sh
+path/to/paftools.js             # only if k8 is on your $PATH
+k8 path/to/paftools.js
+```
+
+In a nutshell, paftools.js has the following commands:
+
+```
+Usage: paftools.js <command> [arguments]
+Commands:
+  view       convert PAF to BLAST-like (for eyeballing) or MAF
+  splice2bed convert spliced alignment in PAF/SAM to BED12
+  sam2paf    convert SAM to PAF
+  delta2paf  convert MUMmer's delta to PAF
+  gff2bed    convert GTF/GFF3 to BED12
+
+  stat       collect basic mapping information in PAF/SAM
+  liftover   simplistic liftOver
+  call       call variants from asm-to-ref alignment with the cs tag
+  bedcov     compute the number of bases covered
+
+  mapeval    evaluate mapping accuracy using mason2/PBSIM-simulated FASTQ
+  mason2fq   convert mason2-simulated SAM to FASTQ
+  pbsim2fq   convert PBSIM-simulated MAF to FASTQ
+  junceval   evaluate splice junction consistency with known annotations
+  ov-eval    evaluate read overlap sensitivity using read-to-ref mapping
+```
+
+paftools.js seamlessly reads both plain text files and gzip'd text files.
+
+## <a name="eval"></a>Evaluation
+
+### <a name="mapeval"></a>Evaluating mapping accuracy with simulated reads
+
+The **pbsim2fq** command of paftools.js converts the MAF output of [pbsim][pbsim]
+to FASTQ and encodes the true mapping position in the read name in a format like
+`S1_33!chr1!225258409!225267761!-`. Similarly, the **mason2fq** command
+converts [mason2][mason2] simulated SAM to FASTQ.
+
+Command **mapeval** evaluates mapped SAM/PAF. Here is example output:
+
+```
+Q       60      32478   0       0.000000000     32478
+Q       22      16      1       0.000030775     32494
+Q       21      43      1       0.000061468     32537
+Q       19      73      1       0.000091996     32610
+Q       14      66      1       0.000122414     32676
+Q       10      27      3       0.000214048     32703
+Q       8       14      1       0.000244521     32717
+Q       7       13      2       0.000305530     32730
+Q       6       46      1       0.000335611     32776
+Q       3       10      1       0.000366010     32786
+Q       2       20      2       0.000426751     32806
+Q       1       248     94      0.003267381     33054
+Q       0       31      17      0.003778147     33085
+U       3
+```
+
+where each Q-line gives the quality threshold, the number of reads mapped with
+mapping quality equal to or greater than the threshold, number of wrong
+mappings, accumulative mapping error rate and the accumulative number of
+mapped reads. The U-line, if present, gives the number of unmapped reads if
+they are present in the SAM file.
+
+Suppose the reported mapping coordinate overlap with the true coordinate like
+the following:
+
+```
+truth:   --------------------
+mapper:           ----------------------
+         |<- l1 ->|<-- o -->|<-- l2 -->|
+```
+
+Let `r=o/(l1+o+l2)`. The reported mapping is considered correct if `r>0.1` by
+default.
+
+### <a name="oveval"></a>Evaluating read overlap sensitivity
+
+Command **ov-eval** takes *sorted* read-to-reference alignment and read
+overlaps in PAF as input, and evaluates the sensitivity. For example:
+
+```sh
+minimap2 -cx map-pb ref.fa reads.fq.gz | sort -k6,6 -k8,8n > reads-to-ref.paf
+minimap2 -x ava-pb reads.fq.gz reads.fq.gz > ovlp.paf
+k8 ov-eval.js reads-to-ref.paf ovlp.paf
+```
+
+## <a name="asmvar"></a>Calling Variants from Haploid Assemblies
+
+The **call** command of paftools.js calls variants from coordinate-sorted
+assembly-to-reference alignment. It calls variants from the [cs tag][cs] and
+identifies confident/callable regions as those covered by exactly one contig.
+Here are example command lines:
+
+```sh
+minimap2 -cx asm5 -t8 --cs ref.fa asm.fa > asm.paf  # keeping this file is recommended; --cs required!
+sort -k6,6 -k8,8n asm.paf > asm.srt.paf             # sort by reference start coordinate
+k8 paftools.js call asm.srt.paf > asm.var.txt
+```
+
+Here is sample output:
+
+```
+V   chr1    2276040 2276041 1   60  c   g   LJII01000171.1  1217409 1217410 +
+V   chr1    2280409 2280410 1   60  a   g   LJII01000171.1  1221778 1221779 +
+V   chr1    2280504 2280505 1   60  a   g   LJII01000171.1  1221873 1221874 +
+R   chr1    2325140 2436340
+V   chr1    2325287 2325287 1   60  -   ct  LJII01000171.1  1272894 1272896 +
+V   chr1    2325642 2325644 1   60  tt  -   LJII01000171.1  1273251 1273251 +
+V   chr1    2326051 2326052 1   60  c   t   LJII01000171.1  1273658 1273659 +
+V   chr1    2326287 2326288 1   60  c   t   LJII01000171.1  1273894 1273895 +
+```
+
+where a line starting with `R` gives regions covered by one query contig, and a
+V-line encodes a variant in the following format: chr, start, end, query depth,
+mapping quality, REF allele, ALT allele, query name, query start, end and the
+query orientation. Generally, you should only look at variants where column 5
+is one.
+
+By default, when calling variants, "paftools.js call" ignores alignments 50kb
+or shorter; when deriving callable regions, it ignores alignments 10kb or
+shorter.  It uses two thresholds to avoid edge effects. These defaults are
+designed for long-read assemblies. For short reads, both should be reduced.
+
+
+
+[paf]: https://github.com/lh3/miniasm/blob/master/PAF.md
+[cs]: https://github.com/lh3/minimap2#cs
 [k8]: https://github.com/attractivechaos/k8
-[k8bin]: https://github.com/attractivechaos/k8/releases
 [maf]: https://genome.ucsc.edu/FAQ/FAQformat#format5
 [pbsim]: https://github.com/pfaucon/PBSIM-PacBio-Simulator
+[mason2]: https://github.com/seqan/seqan/tree/master/apps/mason2

misc/intron-eval.js

@@ -1,266 +0,0 @@
-/*******************************
- * Command line option parsing *
- *******************************/
-
-var getopt = function(args, ostr) {
-	var oli; // option letter list index
-	if (typeof(getopt.place) == 'undefined')
-		getopt.ind = 0, getopt.arg = null, getopt.place = -1;
-	if (getopt.place == -1) { // update scanning pointer
-		if (getopt.ind >= args.length || args[getopt.ind].charAt(getopt.place = 0) != '-') {
-			getopt.place = -1;
-			return null;
-		}
-		if (getopt.place + 1 < args[getopt.ind].length && args[getopt.ind].charAt(++getopt.place) == '-') { // found "--"
-			++getopt.ind;
-			getopt.place = -1;
-			return null;
-		}
-	}
-	var optopt = args[getopt.ind].charAt(getopt.place++); // character checked for validity
-	if (optopt == ':' || (oli = ostr.indexOf(optopt)) < 0) {
-		if (optopt == '-') return null; //  if the user didn't specify '-' as an option, assume it means null.
-		if (getopt.place < 0) ++getopt.ind;
-		return '?';
-	}
-	if (oli+1 >= ostr.length || ostr.charAt(++oli) != ':') { // don't need argument
-		getopt.arg = null;
-		if (getopt.place < 0 || getopt.place >= args[getopt.ind].length) ++getopt.ind, getopt.place = -1;
-	} else { // need an argument
-		if (getopt.place >= 0 && getopt.place < args[getopt.ind].length)
-			getopt.arg = args[getopt.ind].substr(getopt.place);
-		else if (args.length <= ++getopt.ind) { // no arg
-			getopt.place = -1;
-			if (ostr.length > 0 && ostr.charAt(0) == ':') return ':';
-			return '?';
-		} else getopt.arg = args[getopt.ind]; // white space
-		getopt.place = -1;
-		++getopt.ind;
-	}
-	return optopt;
-}
-
-/***********************
- * Interval operations *
- ***********************/
-
-Interval = {};
-
-Interval.sort = function(a)
-{
-	if (typeof a[0] == 'number')
-		a.sort(function(x, y) { return x - y });
-	else a.sort(function(x, y) { return x[0] != y[0]? x[0] - y[0] : x[1] - y[1] });
-}
-
-Interval.merge = function(a, sorted)
-{
-	if (typeof sorted == 'undefined') sorted = true;
-	if (!sorted) Interval.sort(a);
-	var k = 0;
-	for (var i = 1; i < a.length; ++i) {
-		if (a[k][1] >= a[i][0])
-			a[k][1] = a[k][1] > a[i][1]? a[k][1] : a[i][1];
-		else a[++k] = a[i].slice(0);
-	}
-	a.length = k + 1;
-}
-
-Interval.index_end = function(a, sorted)
-{
-	if (a.length == 0) return;
-	if (typeof sorted == 'undefined') sorted = true;
-	if (!sorted) Interval.sort(a);
-	a[0].push(0);
-	var k = 0, k_en = a[0][1];
-	for (var i = 1; i < a.length; ++i) {
-		if (k_en <= a[i][0]) {
-			for (++k; k < i; ++k)
-				if (a[k][1] > a[i][0])
-					break;
-			k_en = a[k][1];
-		}
-		a[i].push(k);
-	}
-}
-
-Interval.find_intv = function(a, x)
-{
-	var left = -1, right = a.length;
-	if (typeof a[0] == 'number') {
-		while (right - left > 1) {
-			var mid = left + ((right - left) >> 1);
-			if (a[mid] > x) right = mid;
-			else if (a[mid] < x) left = mid;
-			else return mid;
-		}
-	} else {
-		while (right - left > 1) {
-			var mid = left + ((right - left) >> 1);
-			if (a[mid][0] > x) right = mid;
-			else if (a[mid][0] < x) left = mid;
-			else return mid;
-		}
-	}
-	return left;
-}
-
-Interval.find_ovlp = function(a, st, en)
-{
-	if (a.length == 0 || st >= en) return [];
-	var l = Interval.find_intv(a, st);
-	var k = l < 0? 0 : a[l][a[l].length - 1];
-	var b = [];
-	for (var i = k; i < a.length; ++i) {
-		if (a[i][0] >= en) break;
-		else if (st < a[i][1])
-			b.push(a[i]);
-	}
-	return b;
-}
-
-/*****************
- * Main function *
- *****************/
-
-var c, l_fuzzy = 0, print_ovlp = false, print_err_only = false, first_only = false;
-while ((c = getopt(arguments, "l:ep")) != null) {
-	if (c == 'l') l_fuzzy = parseInt(getopt.arg);
-	else if (c == 'e') print_err_only = print_ovlp = true;
-	else if (c == 'p') print_ovlp = true;
-}
-
-if (arguments.length - getopt.ind < 2) {
-	print("Usage: k8 intron-eval.js [options] <gene.gtf> <aln.sam>");
-	exit(1);
-}
-
-var file, buf = new Bytes();
-
-var tr = {};
-file = new File(arguments[getopt.ind]);
-while (file.readline(buf) >= 0) {
-	var m, t = buf.toString().split("\t");
-	if (t[0].charAt(0) == '#') continue;
-	if (t[2] != 'exon') continue;
-	var st = parseInt(t[3]) - 1;
-	var en = parseInt(t[4]);
-	if ((m = /transcript_id "(\S+)"/.exec(t[8])) == null) continue;
-	var tid = m[1];
-	if (tr[tid] == null) tr[tid] = [t[0], t[6], 0, 0, []];
-	tr[tid][4].push([st, en]);
-}
-file.close();
-
-var anno = {};
-for (var tid in tr) {
-	var t = tr[tid];
-	Interval.sort(t[4]);
-	t[2] = t[4][0][0];
-	t[3] = t[4][t[4].length - 1][1];
-	if (anno[t[0]] == null) anno[t[0]] = [];
-	var s = t[4];
-	for (var i = 0; i < s.length - 1; ++i) {
-		if (s[i][1] >= s[i+1][0]) throw Error("ERROR: wrong annotation!");
-		anno[t[0]].push([s[i][1], s[i+1][0]]);
-	}
-}
-tr = null;
-
-for (var chr in anno) {
-	var e = anno[chr];
-	if (e.length == 0) continue;
-	Interval.sort(e);
-	var k = 0;
-	for (var i = 1; i < e.length; ++i) // dedup
-		if (e[i][0] != e[k][0] || e[i][1] != e[k][1])
-			e[++k] = e[i].slice(0);
-	e.length = k + 1;
-	Interval.index_end(e);
-}
-
-var n_pri = 0, n_unmapped = 0, n_mapped = 0;
-var n_sgl = 0, n_splice = 0, n_splice_hit = 0, n_splice_novel = 0;
-
-file = new File(arguments[getopt.ind+1]);
-var last_qname = null;
-var re_cigar = /(\d+)([MIDNSHX=])/g;
-while (file.readline(buf) >= 0) {
-	var m, t = buf.toString().split("\t");
-
-	if (t[0].charAt(0) == '@') continue;
-	var flag = parseInt(t[1]);
-	if (flag&0x100) continue;
-	if (first_only && last_qname == t[0]) continue;
-	if (t[2] == '*') {
-		++n_unmapped;
-		continue;
-	} else {
-		++n_pri;
-		if (last_qname != t[0]) ++n_mapped;
-	}
-
-	var pos = parseInt(t[3]) - 1, intron = [];
-	while ((m = re_cigar.exec(t[5])) != null) {
-		var len = parseInt(m[1]), op = m[2];
-		if (op == 'N') {
-			intron.push([pos, pos + len]);
-			pos += len;
-		} else if (op == 'M' || op == 'X' || op == '=' || op == 'D') pos += len;
-	}
-	if (intron.length == 0) {
-		++n_sgl;
-		continue;
-	}
-	n_splice += intron.length;
-
-	var chr = anno[t[2]];
-	if (chr != null) {
-		for (var i = 0; i < intron.length; ++i) {
-			var o = Interval.find_ovlp(chr, intron[i][0], intron[i][1]);
-			if (o.length > 0) {
-				var hit = false;
-				for (var j = 0; j < o.length; ++j) {
-					var st_diff = intron[i][0] - o[j][0];
-					var en_diff = intron[i][1] - o[j][1];
-					if (st_diff < 0) st_diff = -st_diff;
-					if (en_diff < 0) en_diff = -en_diff;
-					if (st_diff <= l_fuzzy && en_diff <= l_fuzzy)
-						++n_splice_hit, hit = true;
-					if (hit) break;
-				}
-				if (print_ovlp) {
-					var type = hit? 'C' : 'P';
-					if (hit && print_err_only) continue;
-					var x = '[';
-					for (var j = 0; j < o.length; ++j) {
-						if (j) x += ', ';
-						x += '(' + o[j][0] + "," + o[j][1] + ')';
-					}
-					x += ']';
-					print(type, t[0], i+1, t[2], intron[i][0], intron[i][1], x);
-				}
-			} else {
-				++n_splice_novel;
-				if (print_ovlp)
-					print('N', t[0], i+1, t[2], intron[i][0], intron[i][1]);
-			}
-		}
-	} else {
-		n_splice_novel += intron.length;
-	}
-	last_qname = t[0];
-}
-file.close();
-
-buf.destroy();
-
-if (!print_ovlp) {
-	print("# unmapped reads: " + n_unmapped);
-	print("# mapped reads: " + n_mapped);
-	print("# primary alignments: " + n_pri);
-	print("# singletons: " + n_sgl);
-	print("# predicted introns: " + n_splice);
-	print("# non-overlapping introns: " + n_splice_novel);
-	print("# correct introns: " + n_splice_hit + " (" + (n_splice_hit / n_splice * 100).toFixed(2) + "%)");
-}

misc/mapstat.js

@@ -1,183 +0,0 @@
-var getopt = function(args, ostr) {
-	var oli; // option letter list index
-	if (typeof(getopt.place) == 'undefined')
-		getopt.ind = 0, getopt.arg = null, getopt.place = -1;
-	if (getopt.place == -1) { // update scanning pointer
-		if (getopt.ind >= args.length || args[getopt.ind].charAt(getopt.place = 0) != '-') {
-			getopt.place = -1;
-			return null;
-		}
-		if (getopt.place + 1 < args[getopt.ind].length && args[getopt.ind].charAt(++getopt.place) == '-') { // found "--"
-			++getopt.ind;
-			getopt.place = -1;
-			return null;
-		}
-	}
-	var optopt = args[getopt.ind].charAt(getopt.place++); // character checked for validity
-	if (optopt == ':' || (oli = ostr.indexOf(optopt)) < 0) {
-		if (optopt == '-') return null; //  if the user didn't specify '-' as an option, assume it means null.
-		if (getopt.place < 0) ++getopt.ind;
-		return '?';
-	}
-	if (oli+1 >= ostr.length || ostr.charAt(++oli) != ':') { // don't need argument
-		getopt.arg = null;
-		if (getopt.place < 0 || getopt.place >= args[getopt.ind].length) ++getopt.ind, getopt.place = -1;
-	} else { // need an argument
-		if (getopt.place >= 0 && getopt.place < args[getopt.ind].length)
-			getopt.arg = args[getopt.ind].substr(getopt.place);
-		else if (args.length <= ++getopt.ind) { // no arg
-			getopt.place = -1;
-			if (ostr.length > 0 && ostr.charAt(0) == ':') return ':';
-			return '?';
-		} else getopt.arg = args[getopt.ind]; // white space
-		getopt.place = -1;
-		++getopt.ind;
-	}
-	return optopt;
-}
-
-var c, gap_out_len = null;
-while ((c = getopt(arguments, "l:")) != null)
-	if (c == 'l') gap_out_len = parseInt(getopt.arg);
-
-if (getopt.ind == arguments.length) {
-	print("Usage: k8 mapstat.js [-l gapOutLen] <in.sam>|<in.paf>");
-	exit(1);
-}
-
-var buf = new Bytes();
-var file = new File(arguments[getopt.ind]);
-var re = /(\d+)([MIDSHNX=])/g;
-
-var lineno = 0, n_pri = 0, n_2nd = 0, n_seq = 0, n_cigar_64k = 0, l_tot = 0, l_cov = 0;
-var n_gap = [[0, 0, 0, 0, 0, 0], [0, 0, 0, 0, 0, 0]];
-
-function cov_len(regs)
-{
-	regs.sort(function(a,b) {return a[0]-b[0]});
-	var st = regs[0][0], en = regs[0][1], l = 0;
-	for (var i = 1; i < regs.length; ++i) {
-		if (regs[i][0] < en)
-			en = en > regs[i][1]? en : regs[i][1];
-		else l += en - st, st = regs[i][0], en = regs[i][1];
-	}
-	l += en - st;
-	return l;
-}
-
-var last = null, last_qlen = null, regs = [];
-while (file.readline(buf) >= 0) {
-	var line = buf.toString();
-	++lineno;
-	if (line.charAt(0) != '@') {
-		var t = line.split("\t", 12);
-		var m, rs, cigar = null, is_pri = false, is_sam = false, is_rev = false, tname = null;
-		var atlen = null, aqlen, qs, qe, mapq, ori_qlen;
-		if (t[4] == '+' || t[4] == '-') { // PAF
-			if (!/\ts2:i:\d+/.test(line)) {
-				++n_2nd;
-				continue;
-			}
-			if ((m = /\tcg:Z:(\S+)/.exec(line)) != null)
-				cigar = m[1];
-			if (cigar == null) {
-				warn("WARNING: no CIGAR at line " + lineno);
-				continue;
-			}
-			tname = t[5];
-			qs = parseInt(t[2]), qe = parseInt(t[3]);
-			aqlen = qe - qs;
-			is_rev = t[4] == '+'? false : true;
-			rs = parseInt(t[7]);
-			atlen = parseInt(t[8]) - rs;
-			mapq = parseInt(t[11]);
-			ori_qlen = parseInt(t[1]);
-		} else { // SAM
-			var flag = parseInt(t[1]);
-			if ((flag & 4) || t[2] == '*' || t[5] == '*') continue;
-			if (flag & 0x100) {
-				++n_2nd;
-				continue;
-			}
-			cigar = t[5];
-			tname = t[2];
-			rs = parseInt(t[3]) - 1;
-			mapq = parseInt(t[4]);
-			aqlen = t[9].length;
-			is_sam = true;
-			is_rev = !!(flag&0x10);
-		}
-		++n_pri;
-		if (last != t[0]) {
-			if (last != null) {
-				l_tot += last_qlen;
-				l_cov += cov_len(regs);
-			}
-			regs = [];
-			++n_seq, last = t[0];
-		}
-		var M = 0, tl = 0, ql = 0, clip = [0, 0], n_cigar = 0, sclip = 0;
-		while ((m = re.exec(cigar)) != null) {
-			var l = parseInt(m[1]);
-			++n_cigar;
-			if (m[2] == 'M' || m[2] == '=' || m[2] == 'X') {
-				tl += l, ql += l, M += l;
-			} else if (m[2] == 'I' || m[2] == 'D') {
-				var type;
-				if (l < 50) type = 0;
-				else if (l < 100) type = 1;
-				else if (l < 300) type = 2;
-				else if (l < 400) type = 3;
-				else if (l < 1000) type = 4;
-				else type = 5;
-				if (m[2] == 'I') ql += l, ++n_gap[0][type];
-				else tl += l, ++n_gap[1][type];
-				if (gap_out_len != null && l >= gap_out_len)
-					print(t[0], ql, is_rev? '-' : '+', tname, rs + tl, m[2], l);
-			} else if (m[2] == 'N') {
-				tl += l;
-			} else if (m[2] == 'S') {
-				clip[M == 0? 0 : 1] = l, sclip += l;
-			} else if (m[2] == 'H') {
-				clip[M == 0? 0 : 1] = l;
-			}
-		}
-		if (n_cigar > 65535) ++n_cigar_64k;
-		if (ql + sclip != aqlen)
-			warn("WARNING: aligned query length is inconsistent with CIGAR at line " + lineno + " (" + (ql+sclip) + " != " + aqlen + ")");
-		if (atlen != null && atlen != tl)
-			warn("WARNING: aligned reference length is inconsistent with CIGAR at line " + lineno);
-		if (is_sam) {
-			qs = clip[is_rev? 1 : 0], qe = qs + ql;
-			ori_qlen = clip[0] + ql + clip[1];
-		}
-		regs.push([qs, qe]);
-		last_qlen = ori_qlen;
-	}
-}
-l_tot += last_qlen;
-l_cov += cov_len(regs);
-
-file.close();
-buf.destroy();
-
-if (gap_out_len == null) {
-	print("Number of mapped sequences: " + n_seq);
-	print("Number of primary alignments: " + n_pri);
-	print("Number of secondary alignments: " + n_2nd);
-	print("Number of primary alignments with >65535 CIGAR operations: " + n_cigar_64k);
-	print("Number of bases in mapped sequences: " + l_tot);
-	print("Number of mapped bases: " + l_cov);
-	print("Number of insertions in [0,50): " + n_gap[0][0]);
-	print("Number of insertions in [50,100): " + n_gap[0][1]);
-	print("Number of insertions in [100,300): " + n_gap[0][2]);
-	print("Number of insertions in [300,400): " + n_gap[0][3]);
-	print("Number of insertions in [400,1000): " + n_gap[0][4]);
-	print("Number of insertions in [1000,inf): " + n_gap[0][5]);
-	print("Number of deletions in [0,50): " + n_gap[1][0]);
-	print("Number of deletions in [50,100): " + n_gap[1][1]);
-	print("Number of deletions in [100,300): " + n_gap[1][2]);
-	print("Number of deletions in [300,400): " + n_gap[1][3]);
-	print("Number of deletions in [400,1000): " + n_gap[1][4]);
-	print("Number of deletions in [1000,inf): " + n_gap[1][5]);
-}

misc/paf2aln.js

@@ -1,171 +0,0 @@
-var getopt = function(args, ostr) {
-	var oli; // option letter list index
-	if (typeof(getopt.place) == 'undefined')
-		getopt.ind = 0, getopt.arg = null, getopt.place = -1;
-	if (getopt.place == -1) { // update scanning pointer
-		if (getopt.ind >= args.length || args[getopt.ind].charAt(getopt.place = 0) != '-') {
-			getopt.place = -1;
-			return null;
-		}
-		if (getopt.place + 1 < args[getopt.ind].length && args[getopt.ind].charAt(++getopt.place) == '-') { // found "--"
-			++getopt.ind;
-			getopt.place = -1;
-			return null;
-		}
-	}
-	var optopt = args[getopt.ind].charAt(getopt.place++); // character checked for validity
-	if (optopt == ':' || (oli = ostr.indexOf(optopt)) < 0) {
-		if (optopt == '-') return null; //  if the user didn't specify '-' as an option, assume it means null.
-		if (getopt.place < 0) ++getopt.ind;
-		return '?';
-	}
-	if (oli+1 >= ostr.length || ostr.charAt(++oli) != ':') { // don't need argument
-		getopt.arg = null;
-		if (getopt.place < 0 || getopt.place >= args[getopt.ind].length) ++getopt.ind, getopt.place = -1;
-	} else { // need an argument
-		if (getopt.place >= 0 && getopt.place < args[getopt.ind].length)
-			getopt.arg = args[getopt.ind].substr(getopt.place);
-		else if (args.length <= ++getopt.ind) { // no arg
-			getopt.place = -1;
-			if (ostr.length > 0 && ostr.charAt(0) == ':') return ':';
-			return '?';
-		} else getopt.arg = args[getopt.ind]; // white space
-		getopt.place = -1;
-		++getopt.ind;
-	}
-	return optopt;
-}
-
-var c, maf_out = false, line_len = 80;
-while ((c = getopt(arguments, "ml:")) != null) {
-	if (c == 'm') maf_out = true;
-	else if (c == 'l') line_len = parseInt(getopt.arg); // TODO: not implemented yet
-}
-if (line_len == 0) line_len = 0x7fffffff;
-
-if (getopt.ind == arguments.length) {
-	print("Usage: k8 paf2aln.js [options] <with-cs.paf>");
-	print("Options:");
-	print("  -m        MAF output (BLAST-like output by default)");
-	print("  -l INT    line length in BLAST-like output [80]");
-	print("");
-	print("Note: this script only works when minimap2 is run with option '-S'");
-	exit(1);
-}
-
-function padding_str(x, len, right)
-{
-	var s = x.toString();
-	if (s.length < len) {
-		if (right) s += Array(len - s.length + 1).join(" ");
-		else s = Array(len - s.length + 1).join(" ") + s;
-	}
-	return s;
-}
-
-function update_aln(s_ref, s_qry, s_mid, type, seq, slen)
-{
-	var l = type == '*'? 1 : seq.length;
-	if (type == '=') {
-		s_ref.set(seq);
-		s_qry.set(seq);
-		s_mid.set(Array(l+1).join("|"));
-		slen[0] += l, slen[1] += l;
-	} else if (type == '*') {
-		s_ref.set(seq.charAt(0));
-		s_qry.set(seq.charAt(1));
-		s_mid.set(' ');
-		slen[0] += 1, slen[1] += 1;
-	} else if (type == '+') {
-		s_ref.set(Array(l+1).join("-"));
-		s_qry.set(seq);
-		s_mid.set(Array(l+1).join(" "));
-		slen[1] += l;
-	} else if (type == '-') {
-		s_ref.set(seq);
-		s_qry.set(Array(l+1).join("-"));
-		s_mid.set(Array(l+1).join(" "));
-		slen[0] += l;
-	}
-}
-
-function print_aln(rs, qs, strand, slen, elen, s_ref, s_qry, s_mid)
-{
-	print(["Ref+:", padding_str(rs + slen[0] + 1, 10, false), s_ref.toString(), padding_str(rs + elen[0], 10, true)].join(" "));
-	print("                 " + s_mid.toString());
-	var st, en;
-	if (strand == '+') st = qs + slen[1] + 1, en = qs + elen[1];
-	else st = qs - slen[1], en = qs - elen[1] + 1;
-	print(["Qry" + strand + ":", padding_str(st,               10, false), s_qry.toString(), padding_str(en          , 10, true)].join(" "));
-}
-
-var s_ref = new Bytes(), s_qry = new Bytes(), s_mid = new Bytes();
-var re = /([=\-\+\*])([A-Za-z]+)/g;
-
-var buf = new Bytes();
-var file = new File(arguments[getopt.ind]);
-if (maf_out) print("##maf version=1\n");
-while (file.readline(buf) >= 0) {
-	var m, line = buf.toString();
-	var t = line.split("\t", 12);
-	if ((m = /\tcs:Z:(\S+)/.exec(line)) == null) continue;
-	var cs = m[1];
-	s_ref.length = s_qry.length = s_mid.length = 0;
-	var slen = [0, 0], elen = [0, 0];
-	if (maf_out) {
-		while ((m = re.exec(cs)) != null)
-			update_aln(s_ref, s_qry, s_mid, m[1], m[2], elen);
-		if (maf_out) {
-			var score = (m = /\tAS:i:(\d+)/.exec(line)) != null? parseInt(m[1]) : 0;
-			var len = t[0].length > t[5].length? t[0].length : t[5].length;
-			print("a " + score);
-			print(["s", padding_str(t[5], len, true), padding_str(t[7], 10, false), padding_str(parseInt(t[8]) - parseInt(t[7]), 10, false),
-				   "+", padding_str(t[6], 10, false), s_ref.toString()].join(" "));
-			var qs, qe, ql = parseInt(t[1]);
-			if (t[4] == '+') {
-				qs = parseInt(t[2]);
-				qe = parseInt(t[3]);
-			} else {
-				qs = ql - parseInt(t[3]);
-				qe = ql - parseInt(t[2]);
-			}
-			print(["s", padding_str(t[0], len, true), padding_str(qs, 10, false), padding_str(qe - qs, 10, false),
-				   t[4], padding_str(ql, 10, false), s_qry.toString()].join(" "));
-			print("");
-		}
-	} else {
-		line = line.replace(/\tc[sg]:Z:\S+/g, "");
-		print('>' + line);
-		var rs = parseInt(t[7]), qs = t[4] == '+'? parseInt(t[2]) : parseInt(t[3]);
-		var n_blocks = 0;
-		while ((m = re.exec(cs)) != null) {
-			var start = 0, rest = m[1] == '*'? 1 : m[2].length;
-			while (rest > 0) {
-				var l_proc;
-				if (s_ref.length + rest >= line_len) {
-					l_proc = line_len - s_ref.length;
-					update_aln(s_ref, s_qry, s_mid, m[1], m[1] == '*'? m[2] : m[2].substr(start, l_proc), elen);
-					if (n_blocks > 0) print("");
-					print_aln(rs, qs, t[4], slen, elen, s_ref, s_qry, s_mid);
-					++n_blocks;
-					s_ref.length = s_qry.length = s_mid.length = 0;
-					slen[0] = elen[0], slen[1] = elen[1];
-				} else {
-					l_proc = rest;
-					update_aln(s_ref, s_qry, s_mid, m[1], m[1] == '*'? m[2] : m[2].substr(start, l_proc), elen);
-				}
-				rest -= l_proc, start += l_proc;
-			}
-		}
-		if (s_ref.length > 0) {
-			if (n_blocks > 0) print("");
-			print_aln(rs, qs, t[4], slen, elen, s_ref, s_qry, s_mid);
-			++n_blocks;
-		}
-		print("//");
-	}
-}
-file.close();
-buf.destroy();
-
-s_ref.destroy(); s_qry.destroy(); s_mid.destroy();

misc/sam2paf.js

@@ -1,111 +0,0 @@
-var getopt = function(args, ostr) {
-	var oli; // option letter list index
-	if (typeof(getopt.place) == 'undefined')
-		getopt.ind = 0, getopt.arg = null, getopt.place = -1;
-	if (getopt.place == -1) { // update scanning pointer
-		if (getopt.ind >= args.length || args[getopt.ind].charAt(getopt.place = 0) != '-') {
-			getopt.place = -1;
-			return null;
-		}
-		if (getopt.place + 1 < args[getopt.ind].length && args[getopt.ind].charAt(++getopt.place) == '-') { // found "--"
-			++getopt.ind;
-			getopt.place = -1;
-			return null;
-		}
-	}
-	var optopt = args[getopt.ind].charAt(getopt.place++); // character checked for validity
-	if (optopt == ':' || (oli = ostr.indexOf(optopt)) < 0) {
-		if (optopt == '-') return null; //  if the user didn't specify '-' as an option, assume it means null.
-		if (getopt.place < 0) ++getopt.ind;
-		return '?';
-	}
-	if (oli+1 >= ostr.length || ostr.charAt(++oli) != ':') { // don't need argument
-		getopt.arg = null;
-		if (getopt.place < 0 || getopt.place >= args[getopt.ind].length) ++getopt.ind, getopt.place = -1;
-	} else { // need an argument
-		if (getopt.place >= 0 && getopt.place < args[getopt.ind].length)
-			getopt.arg = args[getopt.ind].substr(getopt.place);
-		else if (args.length <= ++getopt.ind) { // no arg
-			getopt.place = -1;
-			if (ostr.length > 0 && ostr.charAt(0) == ':') return ':';
-			return '?';
-		} else getopt.arg = args[getopt.ind]; // white space
-		getopt.place = -1;
-		++getopt.ind;
-	}
-	return optopt;
-}
-
-var c, pri_only = false;
-while ((c = getopt(arguments, "p")) != null)
-	if (c == 'p') pri_only = true;
-
-var file = arguments.length == getopt.ind? new File() : new File(arguments[getopt.ind]);
-var buf = new Bytes();
-var re = /(\d+)([MIDSHNX=])/g;
-
-var len = {}, lineno = 0;
-while (file.readline(buf) >= 0) {
-	var m, n_cigar = 0, line = buf.toString();
-	++lineno;
-	if (line.charAt(0) == '@') {
-		if (/^@SQ/.test(line)) {
-			var name = (m = /\tSN:(\S+)/.exec(line)) != null? m[1] : null;
-			var l = (m = /\tLN:(\d+)/.exec(line)) != null? parseInt(m[1]) : null;
-			if (name != null && l != null) len[name] = l;
-		}
-		continue;
-	}
-	var t = line.split("\t");
-	var flag = parseInt(t[1]);
-	if (t[9] != '*' && t[10] != '*' && t[9].length != t[10].length) throw Error("ERROR at line " + lineno + ": inconsistent SEQ and QUAL lengths - " + t[9].length + " != " + t[10].length);
-	if (t[2] == '*' || (flag&4)) continue;
-	if (pri_only && (flag&0x100)) continue;
-	var tlen = len[t[2]];
-	if (tlen == null) throw Error("ERROR at line " + lineno + ": can't find the length of contig " + t[2]);
-	var nn = (m = /\tnn:i:(\d+)/.exec(line)) != null? parseInt(m[1]) : 0;
-	var NM = (m = /\tNM:i:(\d+)/.exec(line)) != null? parseInt(m[1]) : null;
-	var have_NM = NM == null? false : true;
-	NM += nn;
-	var clip = [0, 0], I = [0, 0], D = [0, 0], M = 0, N = 0, ql = 0, tl = 0, mm = 0, ext_cigar = false;
-	while ((m = re.exec(t[5])) != null) {
-		var l = parseInt(m[1]);
-		if (m[2] == 'M') M += l, ql += l, tl += l, ext_cigar = false;
-		else if (m[2] == 'I') ++I[0], I[1] += l, ql += l;
-		else if (m[2] == 'D') ++D[0], D[1] += l, tl += l;
-		else if (m[2] == 'N') N += l, tl += l;
-		else if (m[2] == 'S') clip[M == 0? 0 : 1] = l, ql += l;
-		else if (m[2] == 'H') clip[M == 0? 0 : 1] = l;
-		else if (m[2] == '=') M += l, ql += l, tl += l, ext_cigar = true;
-		else if (m[2] == 'X') M += l, ql += l, tl += l, mm += l, ext_cigar = true;
-		++n_cigar;
-	}
-	if (n_cigar > 65535)
-		warn("WARNING at line " + lineno + ": " + n_cigar + " CIGAR operations");
-	if (tl + parseInt(t[3]) - 1 > tlen) {
-		warn("WARNING at line " + lineno + ": alignment end position larger than ref length; skipped");
-		continue;
-	}
-	if (t[9] != '*' && t[9].length != ql) {
-		warn("WARNING at line " + lineno + ": SEQ length inconsistent with CIGAR (" + t[9].length + " != " + ql + "); skipped");
-		continue;
-	}
-	if (!have_NM || ext_cigar) NM = I[1] + D[1] + mm;
-	if (NM < I[1] + D[1] + mm) {
-		warn("WARNING at line " + lineno + ": NM is less than the total number of gaps (" + NM + " < " + (I[1]+D[1]+mm) + ")");
-		NM = I[1] + D[1] + mm;
-	}
-	var extra = ["mm:i:"+(NM-I[1]-D[1]), "io:i:"+I[0], "in:i:"+I[1], "do:i:"+D[0], "dn:i:"+D[1]];
-	var match = M - (NM - I[1] - D[1]);
-	var blen = M + I[1] + D[1];
-	var qlen = M + I[1] + clip[0] + clip[1];
-	var qs, qe;
-	if (flag&16) qs = clip[1], qe = qlen - clip[0];
-	else qs = clip[0], qe = qlen - clip[1];
-	var ts = parseInt(t[3]) - 1, te = ts + M + D[1] + N;
-	var a = [t[0], qlen, qs, qe, flag&16? '-' : '+', t[2], tlen, ts, te, match, blen, t[4]];
-	print(a.join("\t"), extra.join("\t"));
-}
-
-buf.destroy();
-file.close();

misc/sim-eval.js

@@ -1,191 +0,0 @@
-var getopt = function(args, ostr) {
-	var oli; // option letter list index
-	if (typeof(getopt.place) == 'undefined')
-		getopt.ind = 0, getopt.arg = null, getopt.place = -1;
-	if (getopt.place == -1) { // update scanning pointer
-		if (getopt.ind >= args.length || args[getopt.ind].charAt(getopt.place = 0) != '-') {
-			getopt.place = -1;
-			return null;
-		}
-		if (getopt.place + 1 < args[getopt.ind].length && args[getopt.ind].charAt(++getopt.place) == '-') { // found "--"
-			++getopt.ind;
-			getopt.place = -1;
-			return null;
-		}
-	}
-	var optopt = args[getopt.ind].charAt(getopt.place++); // character checked for validity
-	if (optopt == ':' || (oli = ostr.indexOf(optopt)) < 0) {
-		if (optopt == '-') return null; //  if the user didn't specify '-' as an option, assume it means null.
-		if (getopt.place < 0) ++getopt.ind;
-		return '?';
-	}
-	if (oli+1 >= ostr.length || ostr.charAt(++oli) != ':') { // don't need argument
-		getopt.arg = null;
-		if (getopt.place < 0 || getopt.place >= args[getopt.ind].length) ++getopt.ind, getopt.place = -1;
-	} else { // need an argument
-		if (getopt.place >= 0 && getopt.place < args[getopt.ind].length)
-			getopt.arg = args[getopt.ind].substr(getopt.place);
-		else if (args.length <= ++getopt.ind) { // no arg
-			getopt.place = -1;
-			if (ostr.length > 0 && ostr.charAt(0) == ':') return ':';
-			return '?';
-		} else getopt.arg = args[getopt.ind]; // white space
-		getopt.place = -1;
-		++getopt.ind;
-	}
-	return optopt;
-}
-
-var c, max_mapq = 60, mode = 0, err_out_q = 256, print_err = false, ovlp_ratio = 0.1, cap_short_mapq = false;
-while ((c = getopt(arguments, "Q:r:m:c")) != null) {
-	if (c == 'Q') err_out_q = parseInt(getopt.arg), print_err = true;
-	else if (c == 'r') ovlp_ratio = parseFloat(getopt.arg);
-	else if (c == 'm') mode = parseInt(getopt.arg);
-	else if (c == 'c') cap_short_mapq = true;
-}
-
-var file = arguments.length == getopt.ind? new File() : new File(arguments[getopt.ind]);
-var buf = new Bytes();
-
-var tot = [], err = [];
-for (var q = 0; q <= max_mapq; ++q)
-	tot[q] = err[q] = 0;
-
-function is_correct(s, b)
-{
-	if (s[0] != b[0] || s[3] != b[3]) return false;
-	var o, l;
-	if (s[1] < b[1]) {
-		if (s[2] <= b[1]) return false;
-		o = (s[2] < b[2]? s[2] : b[2]) - b[1];
-		l = (s[2] > b[2]? s[2] : b[2]) - s[1];
-	} else {
-		if (b[2] <= s[1]) return false;
-		o = (s[2] < b[2]? s[2] : b[2]) - s[1];
-		l = (s[2] > b[2]? s[2] : b[2]) - b[1];
-	}
-	return o/l > ovlp_ratio? true : false;
-}
-
-function count_err(qname, a, tot, err, mode)
-{
-	if (a.length == 0) return;
-
-	var m, s;
-	if ((m = /^(\S+)!(\S+)!(\d+)!(\d+)!([\+\-])$/.exec(qname)) != null) { // pbsim single-end reads
-		s = [m[1], m[2], parseInt(m[3]), parseInt(m[4]), m[5]];
-	} else if ((m = /^(\S+)!(\S+)!(\d+)_(\d+)!(\d+)_(\d+)!([\+\-])([\+\-])\/([12])$/.exec(qname)) != null) { // mason2 paired-end reads
-		if (m[9] == '1') {
-			s = [m[1], m[2], parseInt(m[3]), parseInt(m[5]), m[7]];
-		} else {
-			s = [m[1], m[2], parseInt(m[4]), parseInt(m[6]), m[8]];
-		}
-	} else throw Error("Failed to parse simulated read names '" + qname + "'");
-	s.shift(); // skip the orginal read name
-
-	if (mode == 0 || mode == 1) { // longest only or first only
-		var max_i = 0;
-		if (mode == 0) { // longest only
-			var max = 0;
-			for (var i = 0; i < a.length; ++i)
-				if (a[i][5] > max)
-					max = a[i][5], max_i = i;
-		}
-		var mapq = a[max_i][4];
-		++tot[mapq];
-		if (!is_correct(s, a[max_i])) {
-			if (mapq >= err_out_q)
-				print('E', qname, a[max_i].join("\t"));
-			++err[mapq];
-		}
-	} else if (mode == 2) { // all primary mode
-		var max_err_mapq = -1, max_mapq = 0, max_err_i = -1;
-		if (cap_short_mapq) {
-			var max = 0, max_q = 0;
-			for (var i = 0; i < a.length; ++i)
-				if (a[i][5] > max)
-					max = a[i][5], max_q = a[i][4];
-			for (var i = 0; i < a.length; ++i)
-				a[i][4] = max_q < a[i][4]? max_q : a[i][4];
-		}
-		for (var i = 0; i < a.length; ++i) {
-			max_mapq = max_mapq > a[i][4]? max_mapq : a[i][4];
-			if (!is_correct(s, a[i]))
-				if (a[i][4] > max_err_mapq)
-					max_err_mapq = a[i][4], max_err_i = i;
-		}
-		if (max_err_mapq >= 0) {
-			++tot[max_err_mapq], ++err[max_err_mapq];
-			if (max_err_mapq >= err_out_q)
-				print('E', qname, a[max_err_i].join("\t"));
-		} else ++tot[max_mapq];
-	}
-}
-
-var lineno = 0, last = null, a = [], n_unmapped = null;
-var re_cigar = /(\d+)([MIDSHN])/g;
-while (file.readline(buf) >= 0) {
-	var m, line = buf.toString();
-	++lineno;
-	if (line[0] != '@') {
-		var t = line.split("\t");
-		if (t[4] == '+' || t[4] == '-') { // PAF
-			if (last != t[0]) {
-				if (last != null) count_err(last, a, tot, err, mode);
-				a = [], last = t[0];
-			}
-			if (/\ts1:i:\d+/.test(line) && !/\ts2:i:\d+/.test(line)) // secondary alignment in minimap2 PAF
-				continue;
-			var mapq = parseInt(t[11]);
-			if (mapq > max_mapq) mapq = max_mapq;
-			a.push([t[5], parseInt(t[7]), parseInt(t[8]), t[4], mapq, parseInt(t[9])]);
-		} else { // SAM
-			var flag = parseInt(t[1]);
-			var read_no = flag>>6&0x3;
-			var qname = read_no == 1 || read_no == 2? t[0] + '/' + read_no : t[0];
-			if (last != qname) {
-				if (last != null) count_err(last, a, tot, err, mode);
-				a = [], last = qname;
-			}
-			if (flag&0x100) continue; // secondary alignment
-			if ((flag&0x4) || t[2] == '*') { // unmapped
-				if (n_unmapped == null) n_unmapped = 0;
-				++n_unmapped;
-				continue;
-			}
-			var mapq = parseInt(t[4]);
-			if (mapq > max_mapq) mapq = max_mapq;
-			var pos = parseInt(t[3]) - 1, pos_end = pos;
-			var n_gap = 0, mlen = 0;
-			while ((m = re_cigar.exec(t[5])) != null) {
-				var len = parseInt(m[1]);
-				if (m[2] == 'M') pos_end += len, mlen += len;
-				else if (m[2] == 'I') n_gap += len;
-				else if (m[2] == 'D') n_gap += len, pos_end += len;
-			}
-			var score = pos_end - pos;
-			if ((m = /\tNM:i:(\d+)/.exec(line)) != null) {
-				var NM = parseInt(m[1]);
-				if (NM >= n_gap) score = mlen - (NM - n_gap);
-			}
-			a.push([t[2], pos, pos_end, (flag&16)? '-' : '+', mapq, score]);
-		}
-	}
-}
-if (last != null) count_err(last, a, tot, err, mode);
-
-buf.destroy();
-file.close();
-
-var sum_tot = 0, sum_err = 0, q_out = -1, sum_tot2 = 0, sum_err2 = 0;
-for (var q = max_mapq; q >= 0; --q) {
-	if (tot[q] == 0) continue;
-	if (q_out < 0 || err[q] > 0) {
-		if (q_out >= 0) print('Q', q_out, sum_tot, sum_err, (sum_err2/sum_tot2).toFixed(9));
-		sum_tot = sum_err = 0, q_out = q;
-	}
-	sum_tot += tot[q], sum_err += err[q];
-	sum_tot2 += tot[q], sum_err2 += err[q];
-}
-print('Q', q_out, sum_tot, sum_err, (sum_err2/sum_tot2).toFixed(9));
-if (n_unmapped != null) print('U', n_unmapped);

misc/sim-mason2.js

@@ -1,105 +0,0 @@
-Bytes.prototype.reverse = function()
-{
-	for (var i = 0; i < this.length>>1; ++i) {
-		var tmp = this[i];
-		this[i] = this[this.length - i - 1];
-		this[this.length - i - 1] = tmp;
-	}
-}
-
-// reverse complement a DNA string
-Bytes.prototype.revcomp = function()
-{
-	if (Bytes.rctab == null) {
-		var s1 = 'WSATUGCYRKMBDHVNwsatugcyrkmbdhvn';
-		var s2 = 'WSTAACGRYMKVHDBNwstaacgrymkvhdbn';
-		Bytes.rctab = [];
-		for (var i = 0; i < 256; ++i) Bytes.rctab[i] = 0;
-		for (var i = 0; i < s1.length; ++i)
-			Bytes.rctab[s1.charCodeAt(i)] = s2.charCodeAt(i);
-	}
-	for (var i = 0; i < this.length>>1; ++i) {
-		var tmp = this[this.length - i - 1];
-		this[this.length - i - 1] = Bytes.rctab[this[i]];
-		this[i] = Bytes.rctab[tmp];
-	}
-	if (this.length&1)
-		this[this.length>>1] = Bytes.rctab[this[this.length>>1]];
-}
-
-if (arguments.length == 0) {
-	print("Usage: k8 sim-mason2.js <mason.sam>");
-	exit(1);
-}
-
-function print_se(a)
-{
-	print('@' + a.slice(0, 5).join("!") + " " + a[8]);
-	print(a[5]);
-	print("+");
-	print(a[6]);
-}
-
-var buf = new Bytes(), buf2 = new Bytes();
-var file = new File(arguments[0]);
-var re = /(\d+)([MIDSHN])/g;
-var last = null;
-while (file.readline(buf) >= 0) {
-	var t = buf.toString().split("\t");
-	if (t[0].charAt(0) == '@') continue;
-	var m, l_ref = 0;
-	while ((m = re.exec(t[5])) != null)
-		if (m[2] == 'D' || m[2] == 'M' || m[2] == 'N')
-			l_ref += parseInt(m[1]);
-	var flag = parseInt(t[1]);
-	var rev = !!(flag&16);
-	var seq, qual;
-	if (rev) {
-		buf2.length = 0;
-		buf2.set(t[9], 0);
-		buf2.revcomp();
-		seq = buf2.toString();
-		buf2.set(t[10], 0);
-		buf2.reverse();
-		qual = buf2.toString();
-	} else seq = t[9], qual = t[10];
-	var qname = t[0];
-	qname = qname.replace(/^simulated./, "");
-	var chr = t[2];
-	var pos = parseInt(t[3]) - 1;
-	var strand = (flag&16)? '-' : '+';
-	var read_no = flag&0xc0;
-	if (read_no == 0x40) read_no = 1;
-	else if (read_no == 0x80) read_no = 2;
-	else read_no = 0;
-	var err = 0, snp = 0, indel = 0;
-	for (var i = 11; i < t.length; ++i) {
-		if ((m = /^XE:i:(\d+)/.exec(t[i])) != null) err = m[1];
-		else if ((m = /^XS:i:(\d+)/.exec(t[i])) != null) snp = m[1];
-		else if ((m = /^XI:i:(\d+)/.exec(t[i])) != null) indel = m[1];
-	}
-	var comment = [err, snp, indel].join(":");
-	if (last == null) {
-		last = [qname, chr, pos, pos + l_ref, strand, seq, qual, read_no, comment];
-	} else if (last[0] != qname) {
-		print_se(last);
-		last = [qname, chr, pos, pos + l_ref, strand, seq, qual, read_no, comment];
-	} else {
-		if (read_no == 2) { // last[] is the first read
-			if (last[7] != 1) throw Error("ERROR: can't find read1");
-			var name = [qname, chr, last[2] + "_" + pos, last[3] + "_" + (pos + l_ref), last[4] + strand].join("!");
-			print('@' + name + '/1' + ' ' + last[8]); print(last[5]); print("+"); print(last[6]);
-			print('@' + name + '/2' + ' ' + comment); print(seq); print("+"); print(qual);
-		} else {
-			if (last[7] != 2) throw Error("ERROR: can't find read2");
-			var name = [qname, chr, pos + "_" + last[2], (pos + l_ref) + "_" + last[3], strand + last[4]].join("!");
-			print('@' + name + '/1' + ' ' + comment); print(seq); print("+"); print(qual);
-			print('@' + name + '/2' + ' ' + last[8]); print(last[5]); print("+"); print(last[6]);
-		}
-		last = null;
-	}
-}
-if (last != null) print_se(last);
-file.close();
-buf.destroy();
-buf2.destroy();

misc/sim-pbsim.js

@@ -1,81 +0,0 @@
-Bytes.prototype.reverse = function()
-{
-	for (var i = 0; i < this.length>>1; ++i) {
-		var tmp = this[i];
-		this[i] = this[this.length - i - 1];
-		this[this.length - i - 1] = tmp;
-	}
-}
-
-// reverse complement a DNA string
-Bytes.prototype.revcomp = function()
-{
-	if (Bytes.rctab == null) {
-		var s1 = 'WSATUGCYRKMBDHVNwsatugcyrkmbdhvn';
-		var s2 = 'WSTAACGRYMKVHDBNwstaacgrymkvhdbn';
-		Bytes.rctab = [];
-		for (var i = 0; i < 256; ++i) Bytes.rctab[i] = 0;
-		for (var i = 0; i < s1.length; ++i)
-			Bytes.rctab[s1.charCodeAt(i)] = s2.charCodeAt(i);
-	}
-	for (var i = 0; i < this.length>>1; ++i) {
-		var tmp = this[this.length - i - 1];
-		this[this.length - i - 1] = Bytes.rctab[this[i]];
-		this[i] = Bytes.rctab[tmp];
-	}
-	if (this.length&1)
-		this[this.length>>1] = Bytes.rctab[this[this.length>>1]];
-}
-
-if (arguments.length < 2) {
-	print("Usage: k8 sim-pbsim.js <ref.fa.fai> <pbsim1.maf> [[pbsim2.maf] ...]");
-	exit(1);
-}
-
-var file, buf = new Bytes(), buf2 = new Bytes();
-file = new File(arguments[0]);
-var chr_list = [];
-while (file.readline(buf) >= 0) {
-	var t = buf.toString().split(/\s+/);
-	chr_list.push(t[0]);
-}
-file.close();
-
-for (var k = 1; k < arguments.length; ++k) {
-	var fn = arguments[k];
-	file = new File(fn);
-	var state = 0, reg;
-	while (file.readline(buf) >= 0) {
-		var line = buf.toString();
-		if (state == 0 && line.charAt(0) == 'a') {
-			state = 1;
-		} else if (state == 1 && line.charAt(0) == 's') {
-			var t = line.split(/\s+/);
-			var st = parseInt(t[2]);
-			reg = [st, st + parseInt(t[3])];
-			state = 2;
-		} else if (state == 2 && line.charAt(0) == 's') {
-			var m, t = line.split(/\s+/);
-			if ((m = /S(\d+)_\d+/.exec(t[1])) == null) throw Error("Failed to parse the read name");
-			var chr_id = parseInt(m[1]) - 1;
-			if (chr_id >= chr_list.length) throw Error("Index outside the chr list");
-			var name = [t[1], chr_list[chr_id], reg[0], reg[1], t[4]].join("!");
-			var seq = t[6].replace(/\-/g, "");
-			if (seq.length != parseInt(t[5])) throw Error("Inconsistent read length");
-			if (seq.indexOf("NN") < 0) {
-				if (t[4] == '-') {
-					buf2.set(seq, 0);
-					buf2.length = seq.length;
-					buf2.revcomp();
-					seq = buf2.toString();
-				}
-				print(">" + name);
-				print(seq);
-			}
-			state = 0;
-		}
-	}
-	file.close();
-}
-buf.destroy();
-buf2.destroy();

mmpriv.h

@@ -16,11 +16,21 @@
 #define MM_SEED_LONG_JOIN  (1ULL<<40)
 #define MM_SEED_IGNORE     (1ULL<<41)
 #define MM_SEED_TANDEM     (1ULL<<42)
+#define MM_SEED_SELF       (1ULL<<43)
+
+#define MM_SEED_SEG_SHIFT  48
+#define MM_SEED_SEG_MASK   (0xffULL<<(MM_SEED_SEG_SHIFT))
 
 #ifndef kroundup32
 #define kroundup32(x) (--(x), (x)|=(x)>>1, (x)|=(x)>>2, (x)|=(x)>>4, (x)|=(x)>>8, (x)|=(x)>>16, ++(x))
 #endif
 
+#define mm_seq4_set(s, i, c) ((s)[(i)>>3] |= (uint32_t)(c) << (((i)&7)<<2))
+#define mm_seq4_get(s, i)    ((s)[(i)>>3] >> (((i)&7)<<2) & 0xf)
+
+#define MALLOC(type, len) ((type*)malloc((len) * sizeof(type)))
+#define CALLOC(type, len) ((type*)calloc((len), sizeof(type)))
+
 #ifdef __cplusplus
 extern "C" {
 #endif
@@ -33,30 +43,60 @@ typedef struct __kstring_t {
 } kstring_t;
 #endif
 
+typedef struct {
+	int n_u, n_a;
+	uint64_t *u;
+	mm128_t *a;
+} mm_seg_t;
+
 double cputime(void);
 double realtime(void);
+long peakrss(void);
 
 void radix_sort_128x(mm128_t *beg, mm128_t *end);
 void radix_sort_64(uint64_t *beg, uint64_t *end);
 uint32_t ks_ksmall_uint32_t(size_t n, uint32_t arr[], size_t kk);
 
-void mm_write_sam_SQ(const mm_idx_t *idx);
-void mm_write_sam_hdr_no_SQ(const char *rg, const char *ver, int argc, char *argv[]);
+void mm_sketch(void *km, const char *str, int len, int w, int k, uint32_t rid, int is_hpc, mm128_v *p);
+
+void mm_write_sam_hdr(const mm_idx_t *mi, const char *rg, const char *ver, int argc, char *argv[]);
 void mm_write_paf(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const mm_reg1_t *r, void *km, int opt_flag);
 void mm_write_sam(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, const mm_reg1_t *r, int n_regs, const mm_reg1_t *regs);
-int mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cnt, int min_sc, int is_cdna, int64_t n, mm128_t *a, uint64_t **_u, void *km);
+void mm_write_sam2(kstring_t *s, const mm_idx_t *mi, const mm_bseq1_t *t, int seg_idx, int reg_idx, int n_seg, const int *n_regs, const mm_reg1_t *const* regs, void *km, int opt_flag);
+
+void mm_idxopt_init(mm_idxopt_t *opt);
+const uint64_t *mm_idx_get(const mm_idx_t *mi, uint64_t minier, int *n);
+int32_t mm_idx_cal_max_occ(const mm_idx_t *mi, float f);
+mm128_t *mm_chain_dp(int max_dist_x, int max_dist_y, int bw, int max_skip, int min_cnt, int min_sc, int is_cdna, int n_segs, int64_t n, mm128_t *a, int *n_u_, uint64_t **_u, void *km);
 mm_reg1_t *mm_align_skeleton(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int qlen, const char *qstr, int *n_regs_, mm_reg1_t *regs, mm128_t *a);
 
-mm_reg1_t *mm_gen_regs(void *km, int qlen, int n_u, uint64_t *u, mm128_t *a);
+mm_reg1_t *mm_gen_regs(void *km, uint32_t hash, int qlen, int n_u, uint64_t *u, mm128_t *a);
 void mm_split_reg(mm_reg1_t *r, mm_reg1_t *r2, int n, int qlen, mm128_t *a);
 void mm_sync_regs(void *km, int n_regs, mm_reg1_t *regs);
+int mm_squeeze_a(void *km, int n_regs, mm_reg1_t *regs, mm128_t *a);
 int mm_set_sam_pri(int n, mm_reg1_t *r);
-void mm_set_parent(void *km, float mask_level, int n, mm_reg1_t *r);
-void mm_select_sub(void *km, float mask_level, float pri_ratio, int min_diff, int best_n, int *n_, mm_reg1_t *r);
-void mm_filter_regs(void *km, const mm_mapopt_t *opt, int *n_regs, mm_reg1_t *regs);
+void mm_set_parent(void *km, float mask_level, int n, mm_reg1_t *r, int sub_diff, int hard_mask_level);
+void mm_select_sub(void *km, float pri_ratio, int min_diff, int best_n, int *n_, mm_reg1_t *r);
+void mm_select_sub_multi(void *km, float pri_ratio, float pri1, float pri2, int max_gap_ref, int min_diff, int best_n, int n_segs, const int *qlens, int *n_, mm_reg1_t *r);
+void mm_filter_regs(const mm_mapopt_t *opt, int qlen, int *n_regs, mm_reg1_t *regs);
 void mm_join_long(void *km, const mm_mapopt_t *opt, int qlen, int *n_regs, mm_reg1_t *regs, mm128_t *a);
-void mm_hit_sort_by_dp(void *km, int *n_regs, mm_reg1_t *r);
-void mm_set_mapq(int n_regs, mm_reg1_t *regs, int min_chain_sc);
+void mm_hit_sort(void *km, int *n_regs, mm_reg1_t *r);
+void mm_set_mapq(void *km, int n_regs, mm_reg1_t *regs, int min_chain_sc, int match_sc, int rep_len, int is_sr);
+
+void mm_est_err(const mm_idx_t *mi, int qlen, int n_regs, mm_reg1_t *regs, const mm128_t *a, int32_t n, const uint64_t *mini_pos);
+
+mm_seg_t *mm_seg_gen(void *km, uint32_t hash, int n_segs, const int *qlens, int n_regs0, const mm_reg1_t *regs0, int *n_regs, mm_reg1_t **regs, const mm128_t *a);
+void mm_seg_free(void *km, int n_segs, mm_seg_t *segs);
+void mm_pair(void *km, int max_gap_ref, int dp_bonus, int sub_diff, int match_sc, const int *qlens, int *n_regs, mm_reg1_t **regs);
+
+FILE *mm_split_init(const char *prefix, const mm_idx_t *mi);
+mm_idx_t *mm_split_merge_prep(const char *prefix, int n_splits, FILE **fp, uint32_t *n_seq_part);
+int mm_split_merge(int n_segs, const char **fn, const mm_mapopt_t *opt, int n_split_idx);
+void mm_split_rm_tmp(const char *prefix, int n_splits);
+
+void mm_err_puts(const char *str);
+void mm_err_fwrite(const void *p, size_t size, size_t nitems, FILE *fp);
+void mm_err_fread(void *p, size_t size, size_t nitems, FILE *fp);
 
 #ifdef __cplusplus
 }

options.c

@@ -0,0 +1,183 @@
+#include <stdio.h>
+#include "mmpriv.h"
+
+void mm_idxopt_init(mm_idxopt_t *opt)
+{
+	memset(opt, 0, sizeof(mm_idxopt_t));
+	opt->k = 15, opt->w = 10, opt->flag = 0;
+	opt->bucket_bits = 14;
+	opt->mini_batch_size = 50000000;
+	opt->batch_size = 4000000000ULL;
+}
+
+void mm_mapopt_init(mm_mapopt_t *opt)
+{
+	memset(opt, 0, sizeof(mm_mapopt_t));
+	opt->seed = 11;
+	opt->mid_occ_frac = 2e-4f;
+	opt->sdust_thres = 0; // no SDUST masking
+
+	opt->min_cnt = 3;
+	opt->min_chain_score = 40;
+	opt->bw = 500;
+	opt->max_gap = 5000;
+	opt->max_gap_ref = -1;
+	opt->max_chain_skip = 25;
+
+	opt->mask_level = 0.5f;
+	opt->pri_ratio = 0.8f;
+	opt->best_n = 5;
+
+	opt->max_join_long = 20000;
+	opt->max_join_short = 2000;
+	opt->min_join_flank_sc = 1000;
+	opt->min_join_flank_ratio = 0.5f;
+
+	opt->a = 2, opt->b = 4, opt->q = 4, opt->e = 2, opt->q2 = 24, opt->e2 = 1;
+	opt->sc_ambi = 1;
+	opt->zdrop = 400, opt->zdrop_inv = 200;
+	opt->end_bonus = -1;
+	opt->min_dp_max = opt->min_chain_score * opt->a;
+	opt->min_ksw_len = 200;
+	opt->anchor_ext_len = 20, opt->anchor_ext_shift = 6;
+	opt->max_clip_ratio = 1.0f;
+	opt->mini_batch_size = 500000000;
+
+	opt->pe_ori = 0; // FF
+	opt->pe_bonus = 33;
+}
+
+void mm_mapopt_update(mm_mapopt_t *opt, const mm_idx_t *mi)
+{
+	if ((opt->flag & MM_F_SPLICE_FOR) || (opt->flag & MM_F_SPLICE_REV))
+		opt->flag |= MM_F_SPLICE;
+	if (opt->mid_occ <= 0)
+		opt->mid_occ = mm_idx_cal_max_occ(mi, opt->mid_occ_frac);
+	if (opt->mid_occ < opt->min_mid_occ)
+		opt->mid_occ = opt->min_mid_occ;
+	if (mm_verbose >= 3)
+		fprintf(stderr, "[M::%s::%.3f*%.2f] mid_occ = %d\n", __func__, realtime() - mm_realtime0, cputime() / (realtime() - mm_realtime0), opt->mid_occ);
+}
+
+void mm_mapopt_max_intron_len(mm_mapopt_t *opt, int max_intron_len)
+{
+	if ((opt->flag & MM_F_SPLICE) && max_intron_len > 0)
+		opt->max_gap_ref = opt->bw = max_intron_len;
+}
+
+int mm_set_opt(const char *preset, mm_idxopt_t *io, mm_mapopt_t *mo)
+{
+	if (preset == 0) {
+		mm_idxopt_init(io);
+		mm_mapopt_init(mo);
+	} else if (strcmp(preset, "ava-ont") == 0) {
+		io->flag = 0, io->k = 15, io->w = 5;
+		mo->flag |= MM_F_ALL_CHAINS | MM_F_NO_DIAG | MM_F_NO_DUAL | MM_F_NO_LJOIN;
+		mo->min_chain_score = 100, mo->pri_ratio = 0.0f, mo->max_gap = 10000, mo->max_chain_skip = 25;
+		mo->bw = 2000;
+	} else if (strcmp(preset, "ava-pb") == 0) {
+		io->flag |= MM_I_HPC, io->k = 19, io->w = 5;
+		mo->flag |= MM_F_ALL_CHAINS | MM_F_NO_DIAG | MM_F_NO_DUAL | MM_F_NO_LJOIN;
+		mo->min_chain_score = 100, mo->pri_ratio = 0.0f, mo->max_gap = 10000, mo->max_chain_skip = 25;
+	} else if (strcmp(preset, "map10k") == 0 || strcmp(preset, "map-pb") == 0) {
+		io->flag |= MM_I_HPC, io->k = 19;
+	} else if (strcmp(preset, "map-ont") == 0) {
+		io->flag = 0, io->k = 15;
+	} else if (strcmp(preset, "asm5") == 0) {
+		io->flag = 0, io->k = 19, io->w = 19;
+		mo->a = 1, mo->b = 19, mo->q = 39, mo->q2 = 81, mo->e = 3, mo->e2 = 1, mo->zdrop = mo->zdrop_inv = 200;
+		mo->min_mid_occ = 100;
+		mo->min_dp_max = 200;
+		mo->best_n = 50;
+	} else if (strcmp(preset, "asm10") == 0) {
+		io->flag = 0, io->k = 19, io->w = 19;
+		mo->a = 1, mo->b = 9, mo->q = 16, mo->q2 = 41, mo->e = 2, mo->e2 = 1, mo->zdrop = mo->zdrop_inv = 200;
+		mo->min_mid_occ = 100;
+		mo->min_dp_max = 200;
+		mo->best_n = 50;
+	} else if (strcmp(preset, "asm20") == 0) {
+		io->flag = 0, io->k = 19, io->w = 10;
+		mo->a = 1, mo->b = 4, mo->q = 6, mo->q2 = 26, mo->e = 2, mo->e2 = 1, mo->zdrop = mo->zdrop_inv = 200;
+		mo->min_mid_occ = 100;
+		mo->min_dp_max = 200;
+		mo->best_n = 50;
+	} else if (strcmp(preset, "short") == 0 || strcmp(preset, "sr") == 0) {
+		io->flag = 0, io->k = 21, io->w = 11;
+		mo->flag |= MM_F_SR | MM_F_FRAG_MODE | MM_F_NO_PRINT_2ND | MM_F_2_IO_THREADS | MM_F_HEAP_SORT;
+		mo->pe_ori = 0<<1|1; // FR
+		mo->a = 2, mo->b = 8, mo->q = 12, mo->e = 2, mo->q2 = 24, mo->e2 = 1;
+		mo->zdrop = mo->zdrop_inv = 100;
+		mo->end_bonus = 10;
+		mo->max_frag_len = 800;
+		mo->max_gap = 100;
+		mo->bw = 100;
+		mo->pri_ratio = 0.5f;
+		mo->min_cnt = 2;
+		mo->min_chain_score = 25;
+		mo->min_dp_max = 40;
+		mo->best_n = 20;
+		mo->mid_occ = 1000;
+		mo->max_occ = 5000;
+		mo->mini_batch_size = 50000000;
+	} else if (strcmp(preset, "splice") == 0 || strcmp(preset, "cdna") == 0) {
+		io->flag = 0, io->k = 15, io->w = 5;
+		mo->flag |= MM_F_SPLICE | MM_F_SPLICE_FOR | MM_F_SPLICE_REV | MM_F_SPLICE_FLANK;
+		mo->max_gap = 2000, mo->max_gap_ref = mo->bw = 200000;
+		mo->a = 1, mo->b = 2, mo->q = 2, mo->e = 1, mo->q2 = 32, mo->e2 = 0;
+		mo->noncan = 9;
+		mo->zdrop = 200, mo->zdrop_inv = 100; // because mo->a is halved
+	} else return -1;
+	return 0;
+}
+
+int mm_check_opt(const mm_idxopt_t *io, const mm_mapopt_t *mo)
+{
+	if (mo->split_prefix && (mo->flag & (MM_F_OUT_CS|MM_F_OUT_MD))) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m --cs or --MD doesn't work with --split-prefix\033[0m\n");
+		return -6;
+	}
+	if (io->k <= 0 || io->w <= 0) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m -k and -w must be positive\033[0m\n");
+		return -5;
+	}
+	if (mo->best_n < 0) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m -N must be no less than 0\033[0m\n");
+		return -4;
+	}
+	if (mo->best_n == 0 && mm_verbose >= 2)
+		fprintf(stderr, "[WARNING]\033[1;31m '-N 0' reduces mapping accuracy. Please use '--secondary=no' instead.\033[0m\n");
+	if (mo->pri_ratio < 0.0f || mo->pri_ratio > 1.0f) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m -p must be within 0 and 1 (including 0 and 1)\033[0m\n");
+		return -4;
+	}
+	if ((mo->flag & MM_F_FOR_ONLY) && (mo->flag & MM_F_REV_ONLY)) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m --for-only and --rev-only can't be applied at the same time\033[0m\n");
+		return -3;
+	}
+	if ((mo->q != mo->q2 || mo->e != mo->e2) && !(mo->e > mo->e2 && mo->q + mo->e < mo->q2 + mo->e2)) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m dual gap penalties violating E1>E2 and O1+E1<O2+E2\033[0m\n");
+		return -2;
+	}
+	if ((mo->q + mo->e) + (mo->q2 + mo->e2) > 127) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m scoring system violating ({-O}+{-E})+({-O2}+{-E2}) <= 127\033[0m\n");
+		return -1;
+	}
+	if (mo->zdrop < mo->zdrop_inv) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m Z-drop should not be less than inversion-Z-drop\033[0m\n");
+		return -5;
+	}
+	if ((mo->flag & MM_F_NO_PRINT_2ND) && (mo->flag & MM_F_ALL_CHAINS)) {
+		if (mm_verbose >= 1)
+			fprintf(stderr, "[ERROR]\033[1;31m -X/-P and --secondary=no can't be applied at the same time\033[0m\n");
+		return -5;
+	}
+	return 0;
+}

pe.c

@@ -0,0 +1,177 @@
+#include <stdlib.h>
+#include <math.h>
+#include "mmpriv.h"
+#include "kvec.h"
+
+void mm_select_sub_multi(void *km, float pri_ratio, float pri1, float pri2, int max_gap_ref, int min_diff, int best_n, int n_segs, const int *qlens, int *n_, mm_reg1_t *r)
+{
+	if (pri_ratio > 0.0f && *n_ > 0) {
+		int i, k, n = *n_, n_2nd = 0;
+		int max_dist = n_segs == 2? qlens[0] + qlens[1] + max_gap_ref : 0;
+		for (i = k = 0; i < n; ++i) {
+			int to_keep = 0;
+			if (r[i].parent == i) { // primary
+				to_keep = 1;
+			} else if (r[i].score + min_diff >= r[r[i].parent].score) {
+				to_keep = 1;
+			} else {
+				mm_reg1_t *p = &r[r[i].parent], *q = &r[i];
+				if (p->rev == q->rev && p->rid == q->rid && q->re - p->rs < max_dist && p->re - q->rs < max_dist) { // child and parent are close on the ref
+					if (q->score >= p->score * pri1)
+						to_keep = 1;
+				} else {
+					int is_par_both = (n_segs == 2 && p->qs < qlens[0] && p->qe > qlens[0]);
+					int is_chi_both = (n_segs == 2 && q->qs < qlens[0] && q->qe > qlens[0]);
+					if (is_chi_both || is_chi_both == is_par_both) {
+						if (q->score >= p->score * pri_ratio)
+							to_keep = 1;
+					} else { // the remaining case: is_chi_both == 0 && is_par_both == 1
+						if (q->score >= p->score * pri2)
+							to_keep = 1;
+					}
+				}
+			}
+			if (to_keep && r[i].parent != i) {
+				if (n_2nd++ >= best_n) to_keep = 0; // don't keep if there are too many secondary hits
+			}
+			if (to_keep) r[k++] = r[i];
+			else if (r[i].p) free(r[i].p);
+		}
+		if (k != n) mm_sync_regs(km, k, r); // removing hits requires sync()
+		*n_ = k;
+	}
+}
+
+void mm_set_pe_thru(const int *qlens, int *n_regs, mm_reg1_t **regs)
+{
+	int s, i, n_pri[2], pri[2];
+	n_pri[0] = n_pri[1] = 0;
+	pri[0] = pri[1] = -1;
+	for (s = 0; s < 2; ++s)
+		for (i = 0; i < n_regs[s]; ++i)
+			if (regs[s][i].id == regs[s][i].parent)
+				++n_pri[s], pri[s] = i;
+	if (n_pri[0] == 1 && n_pri[1] == 1) {
+		mm_reg1_t *p = &regs[0][pri[0]];
+		mm_reg1_t *q = &regs[1][pri[1]];
+		if (p->rid == q->rid && p->rev == q->rev && abs(p->rs - q->rs) < 3 && abs(p->re - p->re) < 3
+			&& ((p->qs == 0 && qlens[1] - q->qe == 0) || (q->qs == 0 && qlens[0] - p->qe == 0)))
+		{
+			p->pe_thru = q->pe_thru = 1;
+		}
+	}
+}
+
+#include "ksort.h"
+
+typedef struct {
+	int s, rev;
+	uint64_t key;
+	mm_reg1_t *r;
+} pair_arr_t;
+
+#define sort_key_pair(a) ((a).key)
+KRADIX_SORT_INIT(pair, pair_arr_t, sort_key_pair, 8)
+
+void mm_pair(void *km, int max_gap_ref, int pe_bonus, int sub_diff, int match_sc, const int *qlens, int *n_regs, mm_reg1_t **regs)
+{
+	int i, j, s, n, last[2], dp_thres, segs = 0, max_idx[2];
+	int64_t max;
+	pair_arr_t *a;
+	kvec_t(uint64_t) sc = {0,0,0};
+
+	a = (pair_arr_t*)kmalloc(km, (n_regs[0] + n_regs[1]) * sizeof(pair_arr_t));
+	for (s = n = 0, dp_thres = 0; s < 2; ++s) {
+		int max = 0;
+		for (i = 0; i < n_regs[s]; ++i) {
+			a[n].s = s;
+			a[n].r = &regs[s][i];
+			a[n].rev = a[n].r->rev;
+			a[n].key = (uint64_t)a[n].r->rid << 32 | a[n].r->rs<<1 | (s^a[n].rev);
+			max = max > a[n].r->p->dp_max? max : a[n].r->p->dp_max;
+			++n;
+			segs |= 1<<s;
+		}
+		dp_thres += max;
+	}
+	if (segs != 3) {
+		kfree(km, a); // only one end is mapped
+		return;
+	}
+	dp_thres -= pe_bonus;
+	if (dp_thres < 0) dp_thres = 0;
+	radix_sort_pair(a, a + n);
+
+	max = -1;
+	max_idx[0] = max_idx[1] = -1;
+	last[0] = last[1] = -1;
+	kv_resize(uint64_t, km, sc, (size_t)n);
+	for (i = 0; i < n; ++i) {
+		if (a[i].key & 1) { // reverse first read or forward second read
+			mm_reg1_t *q, *r;
+			if (last[a[i].rev] < 0) continue;
+			r = a[i].r;
+			q = a[last[a[i].rev]].r;
+			if (r->rid != q->rid || r->rs - q->re > max_gap_ref) continue;
+			for (j = last[a[i].rev]; j >= 0; --j) {
+				int64_t score;
+				if (a[j].rev != a[i].rev || a[j].s == a[i].s) continue;
+				q = a[j].r;
+				if (r->rid != q->rid || r->rs - q->re > max_gap_ref) break;
+				if (r->p->dp_max + q->p->dp_max < dp_thres) continue;
+				score = (int64_t)(r->p->dp_max + q->p->dp_max) << 32 | (r->hash + q->hash);
+				if (score > max)
+					max = score, max_idx[a[j].s] = j, max_idx[a[i].s] = i;
+				kv_push(uint64_t, km, sc, score);
+			}
+		} else { // forward first read or reverse second read
+			last[a[i].rev] = i;
+		}
+	}
+	if (sc.n > 1)
+		radix_sort_64(sc.a, sc.a + sc.n);
+
+	if (sc.n > 0 && max > 0) { // found at least one pair
+		int n_sub = 0, mapq_pe;
+		mm_reg1_t *r[2];
+		r[0] = a[max_idx[0]].r, r[1] = a[max_idx[1]].r;
+		r[0]->proper_frag = r[1]->proper_frag = 1;
+		for (s = 0; s < 2; ++s) {
+			if (r[s]->id != r[s]->parent) { // then lift to primary and update parent
+				mm_reg1_t *p = &regs[s][r[s]->parent];
+				for (i = 0; i < n_regs[s]; ++i)
+					if (regs[s][i].parent == p->id)
+						regs[s][i].parent = r[s]->id;
+				p->mapq = 0;
+			}
+			if (!r[s]->sam_pri) { // then sync sam_pri
+				for (i = 0; i < n_regs[s]; ++i)
+					regs[s][i].sam_pri = 0;
+				r[s]->sam_pri = 1;
+			}
+		}
+		mapq_pe = r[0]->mapq > r[1]->mapq? r[0]->mapq : r[1]->mapq;
+		for (i = 0; i < (int)sc.n; ++i)
+			if ((sc.a[i]>>32) + sub_diff >= (uint64_t)max>>32)
+				++n_sub;
+		if (sc.n > 1) {
+			int mapq_pe_alt;
+			mapq_pe_alt = (int)(6.02f * ((max>>32) - (sc.a[sc.n - 2]>>32)) / match_sc - 4.343f * logf(n_sub)); // n_sub > 0 because it counts the optimal, too
+			mapq_pe = mapq_pe < mapq_pe_alt? mapq_pe : mapq_pe_alt;
+		}
+		if (r[0]->mapq < mapq_pe) r[0]->mapq = (int)(.2f * r[0]->mapq + .8f * mapq_pe + .499f);
+		if (r[1]->mapq < mapq_pe) r[1]->mapq = (int)(.2f * r[1]->mapq + .8f * mapq_pe + .499f);
+		if (sc.n == 1) {
+			if (r[0]->mapq < 2) r[0]->mapq = 2;
+			if (r[1]->mapq < 2) r[1]->mapq = 2;
+		} else if ((uint64_t)max>>32 > sc.a[sc.n - 2]>>32) {
+			if (r[0]->mapq < 1) r[0]->mapq = 1;
+			if (r[1]->mapq < 1) r[1]->mapq = 1;
+		}
+	}
+
+	kfree(km, a);
+	kfree(km, sc.a);
+
+	mm_set_pe_thru(qlens, n_regs, regs);
+}

python/README.rst

@@ -0,0 +1,190 @@
+==============================
+Mappy: Minimap2 Python Binding
+==============================
+
+Mappy provides a convenient interface to `minimap2
+<https://github.com/lh3/minimap2>`_, a fast and accurate C program to align
+genomic and transcribe nucleotide sequences.
+
+Installation
+------------
+
+Mappy depends on `zlib <http://zlib.net>`_. It can be installed with `pip
+<https://en.wikipedia.org/wiki/Pip_(package_manager)>`_:
+
+.. code:: shell
+
+	pip install --user mappy
+
+or from the minimap2 github repo (`Cython <http://cython.org>`_ required):
+
+.. code:: shell
+
+	git clone https://github.com/lh3/minimap2
+	cd minimap2
+	python setup.py install
+
+Usage
+-----
+
+The following Python script demonstrates the key functionality of mappy:
+
+.. code:: python
+
+	import mappy as mp
+	a = mp.Aligner("test/MT-human.fa")  # load or build index
+	if not a: raise Exception("ERROR: failed to load/build index")
+	s = a.seq("MT_human", 100, 200)     # retrieve a subsequence from the index
+	print(mp.revcomp(s))                # reverse complement
+	for name, seq, qual in mp.fastx_read("test/MT-orang.fa"): # read a fasta/q sequence
+		for hit in a.map(seq): # traverse alignments
+			print("{}\t{}\t{}\t{}".format(hit.ctg, hit.r_st, hit.r_en, hit.cigar_str))
+
+APIs
+----
+
+Mappy implements two classes and two global function.
+
+Class mappy.Aligner
+~~~~~~~~~~~~~~~~~~~
+
+.. code:: python
+
+	mappy.Aligner(fn_idx_in=None, preset=None, ...)
+
+This constructor accepts the following arguments:
+
+* **fn_idx_in**: index or sequence file name. Minimap2 automatically tests the
+  file type. If a sequence file is provided, minimap2 builds an index. The
+  sequence file can be optionally gzip'd. This option has no effect if **seq**
+  is set.
+
+* **seq**: a single sequence to index. The sequence name will be set to
+  :code:`N/A`.
+
+* **preset**: minimap2 preset. Currently, minimap2 supports the following
+  presets: **sr** for single-end short reads; **map-pb** for PacBio
+  read-to-reference mapping; **map-ont** for Oxford Nanopore read mapping;
+  **splice** for long-read spliced alignment; **asm5** for assembly-to-assembly
+  alignment; **asm10** for full genome alignment of closely related species. Note
+  that the Python module does not support all-vs-all read overlapping.
+
+* **k**: k-mer length, no larger than 28
+
+* **w**: minimizer window size, no larger than 255
+
+* **min_cnt**: mininum number of minimizers on a chain
+
+* **min_chain_score**: minimum chaing score
+
+* **bw**: chaining and alignment band width
+
+* **best_n**: max number of alignments to return
+
+* **n_threads**: number of indexing threads; 3 by default
+
+* **extra_flags**: additional flags defined in minimap.h
+
+* **fn_idx_out**: name of file to which the index is written. This parameter
+  has no effect if **seq** is set.
+
+* **scoring**: scoring system. It is a tuple/list consisting of 4, 6 or 7
+  positive integers. The first 4 elements specify match scoring, mismatch
+  penalty, gap open and gap extension penalty. The 5th and 6th elements, if
+  present, set long-gap open and long-gap extension penalty. The 7th sets a
+  mismatch penalty involving ambiguous bases.
+
+.. code:: python
+
+	mappy.Aligner.map(seq, seq2=None, cs=False, MD=False)
+
+This method aligns :code:`seq` against the index. It is a generator, *yielding*
+a series of :code:`mappy.Alignment` objects. If :code:`seq2` is present, mappy
+performs paired-end alignment, assuming the two ends are in the FR orientation.
+Alignments of the two ends can be distinguished by the :code:`read_num` field
+(see Class mappy.Alignment below). Argument :code:`cs` asks mappy to generate
+the :code:`cs` tag; :code:`MD` is similar. These two arguments might slightly
+degrade performance and are not enabled by default.
+
+.. code:: python
+
+	mappy.Aligner.seq(name, start=0, end=0x7fffffff)
+
+This method retrieves a (sub)sequence from the index and returns it as a Python
+string. :code:`None` is returned if :code:`name` is not present in the index or
+the start/end coordinates are invalid.
+
+Class mappy.Alignment
+~~~~~~~~~~~~~~~~~~~~~
+
+This class describes an alignment. An object of this class has the following
+properties:
+
+* **ctg**: name of the reference sequence the query is mapped to
+
+* **ctg_len**: total length of the reference sequence
+
+* **r_st** and **r_en**: start and end positions on the reference
+
+* **q_st** and **q_en**: start and end positions on the query
+
+* **strand**: +1 if on the forward strand; -1 if on the reverse strand
+
+* **mapq**: mapping quality
+
+* **blen**: length of the alignment, including both alignment matches and gaps
+  but excluding ambiguous bases.
+
+* **mlen**: length of the matching bases in the alignment, excluding ambiguous
+  base matches.
+
+* **NM**: number of mismatches, gaps and ambiguous poistions in the alignment
+
+* **trans_strand**: transcript strand. +1 if on the forward strand; -1 if on the
+  reverse strand; 0 if unknown
+
+* **is_primary**: if the alignment is primary (typically the best and the first
+  to generate)
+
+* **read_num**: read number that the alignment corresponds to; 1 for the first
+  read and 2 for the second read
+
+* **cigar_str**: CIGAR string
+
+* **cigar**: CIGAR returned as an array of shape :code:`(n_cigar,2)`. The two
+  numbers give the length and the operator of each CIGAR operation.
+
+* **MD**: the :code:`MD` tag as in the SAM format. It is an empty string unless
+  the :code:`MD` argument is applied when calling :code:`mappy.Aligner.map()`.
+
+* **cs**: the :code:`cs` tag.
+
+An :code:`Alignment` object can be converted to a string with :code:`str()` in
+the following format:
+
+::
+
+	q_st  q_en  strand  ctg  ctg_len  r_st  r_en  mlen  blen  mapq  cg:Z:cigar_str
+
+It is effectively the PAF format without the QueryName and QueryLength columns
+(the first two columns in PAF).
+
+Miscellaneous Functions
+~~~~~~~~~~~~~~~~~~~~~~~
+
+.. code:: python
+
+	mappy.fastx_read(fn, read_comment=False)
+
+This generator function opens a FASTA/FASTQ file and *yields* a
+:code:`(name,seq,qual)` tuple for each sequence entry. The input file may be
+optionally gzip'd. If :code:`read_comment` is True, this generator yields
+a :code:`(name,seq,qual,comment)` tuple instead.
+
+.. code:: python
+
+	mappy.revcomp(seq)
+
+Return the reverse complement of DNA string :code:`seq`. This function
+recognizes IUB code and preserves the letter cases. Uracil :code:`U` is
+complemented to :code:`A`.

python/cmappy.h

@@ -0,0 +1,152 @@
+#ifndef CMAPPY_H
+#define CMAPPY_H
+
+#include <stdlib.h>
+#include <string.h>
+#include <zlib.h>
+#include "minimap.h"
+#include "kseq.h"
+KSEQ_DECLARE(gzFile)
+
+typedef struct {
+	const char *ctg;
+	int32_t ctg_start, ctg_end;
+	int32_t qry_start, qry_end;
+	int32_t blen, mlen, NM, ctg_len;
+	uint8_t mapq, is_primary;
+	int8_t strand, trans_strand;
+	int32_t seg_id;
+	int32_t n_cigar32;
+	uint32_t *cigar32;
+} mm_hitpy_t;
+
+static inline void mm_reg2hitpy(const mm_idx_t *mi, mm_reg1_t *r, mm_hitpy_t *h)
+{
+	h->ctg = mi->seq[r->rid].name;
+	h->ctg_len = mi->seq[r->rid].len;
+	h->ctg_start = r->rs, h->ctg_end = r->re;
+	h->qry_start = r->qs, h->qry_end = r->qe;
+	h->strand = r->rev? -1 : 1;
+	h->mapq = r->mapq;
+	h->mlen = r->mlen;
+	h->blen = r->blen;
+	h->NM = r->blen - r->mlen + r->p->n_ambi;
+	h->trans_strand = r->p->trans_strand == 1? 1 : r->p->trans_strand == 2? -1 : 0;
+	h->is_primary = (r->id == r->parent);
+	h->seg_id = r->seg_id;
+	h->n_cigar32 = r->p->n_cigar;
+	h->cigar32 = r->p->cigar;
+}
+
+static inline void mm_free_reg1(mm_reg1_t *r)
+{
+	free(r->p);
+}
+
+static inline kseq_t *mm_fastx_open(const char *fn)
+{
+	gzFile fp;
+	fp = fn && strcmp(fn, "-") != 0? gzopen(fn, "r") : gzdopen(fileno(stdin), "r");
+	return kseq_init(fp);
+}
+
+static inline void mm_fastx_close(kseq_t *ks)
+{
+	gzFile fp;
+	fp = ks->f->f;
+	kseq_destroy(ks);
+	gzclose(fp);
+}
+
+static inline int mm_verbose_level(int v)
+{
+	if (v >= 0) mm_verbose = v;
+	return mm_verbose;
+}
+
+static inline void mm_reset_timer(void)
+{
+	extern double realtime(void);
+	mm_realtime0 = realtime();
+}
+
+extern unsigned char seq_comp_table[256];
+static inline mm_reg1_t *mm_map_aux(const mm_idx_t *mi, const char *seq1, const char *seq2, int *n_regs, mm_tbuf_t *b, const mm_mapopt_t *opt)
+{
+	mm_reg1_t *r;
+
+	Py_BEGIN_ALLOW_THREADS
+	if (seq2 == 0) {
+		r = mm_map(mi, strlen(seq1), seq1, n_regs, b, opt, NULL);
+	} else {
+		int _n_regs[2];
+		mm_reg1_t *regs[2];
+		char *seq[2];
+		int i, len[2];
+
+		len[0] = strlen(seq1);
+		len[1] = strlen(seq2);
+		seq[0] = (char*)seq1;
+		seq[1] = strdup(seq2);
+		for (i = 0; i < len[1]>>1; ++i) {
+			int t = seq[1][len[1] - i - 1];
+			seq[1][len[1] - i - 1] = seq_comp_table[(uint8_t)seq[1][i]];
+			seq[1][i] = seq_comp_table[t];
+		}
+		if (len[1]&1) seq[1][len[1]>>1] = seq_comp_table[(uint8_t)seq[1][len[1]>>1]];
+		mm_map_frag(mi, 2, len, (const char**)seq, _n_regs, regs, b, opt, NULL);
+		for (i = 0; i < _n_regs[1]; ++i)
+			regs[1][i].rev = !regs[1][i].rev;
+		*n_regs = _n_regs[0] + _n_regs[1];
+		regs[0] = (mm_reg1_t*)realloc(regs[0], sizeof(mm_reg1_t) * (*n_regs));
+		memcpy(&regs[0][_n_regs[0]], regs[1], _n_regs[1] * sizeof(mm_reg1_t));
+		free(regs[1]);
+		r = regs[0];
+	}
+	Py_END_ALLOW_THREADS
+
+	return r;
+}
+
+static inline char *mappy_revcomp(int len, const uint8_t *seq)
+{
+	int i;
+	char *rev;
+	rev = (char*)malloc(len + 1);
+	for (i = 0; i < len; ++i)
+		rev[len - i - 1] = seq_comp_table[seq[i]];
+	rev[len] = 0;
+	return rev;
+}
+
+static char *mappy_fetch_seq(const mm_idx_t *mi, const char *name, int st, int en, int *len)
+{
+	int i, rid;
+	char *s;
+	*len = 0;
+	rid = mm_idx_name2id(mi, name);
+	if (rid < 0) return 0;
+	if ((uint32_t)st >= mi->seq[rid].len || st >= en) return 0;
+	if (en < 0 || (uint32_t)en > mi->seq[rid].len)
+		en = mi->seq[rid].len;
+	s = (char*)malloc(en - st + 1);
+	*len = mm_idx_getseq(mi, rid, st, en, (uint8_t*)s);
+	for (i = 0; i < *len; ++i)
+		s[i] = "ACGTN"[(uint8_t)s[i]];
+	s[*len] = 0;
+	return s;
+}
+
+static mm_idx_t *mappy_idx_seq(int w, int k, int is_hpc, int bucket_bits, const char *seq, int len)
+{
+	const char *fake_name = "N/A";
+	char *s;
+	mm_idx_t *mi;
+	s = (char*)calloc(len + 1, 1);
+	memcpy(s, seq, len);
+	mi = mm_idx_str(w, k, is_hpc, bucket_bits, 1, (const char**)&s, (const char**)&fake_name);
+	free(s);
+	return mi;
+}
+
+#endif

python/cmappy.pxd

@@ -0,0 +1,133 @@
+from libc.stdint cimport int8_t, uint8_t, int32_t, int64_t, uint32_t, uint64_t
+
+cdef extern from "minimap.h":
+	#
+	# Options
+	#
+	ctypedef struct mm_idxopt_t:
+		short k, w, flag, bucket_bits
+		int mini_batch_size
+		uint64_t batch_size
+
+	ctypedef struct mm_mapopt_t:
+		int seed
+		int sdust_thres
+		int flag
+		int bw
+		int max_gap, max_gap_ref
+		int max_frag_len
+		int max_chain_skip
+		int min_cnt
+		int min_chain_score
+		float mask_level
+		float pri_ratio
+		int best_n
+		int max_join_long, max_join_short
+		int min_join_flank_sc
+		float min_join_flank_ratio;
+		int a, b, q, e, q2, e2
+		int sc_ambi
+		int noncan
+		int zdrop, zdrop_inv
+		int end_bonus
+		int min_dp_max
+		int min_ksw_len
+		int anchor_ext_len, anchor_ext_shift
+		float max_clip_ratio
+		int pe_ori, pe_bonus
+		float mid_occ_frac
+		int32_t min_mid_occ
+		int32_t mid_occ
+		int32_t max_occ
+		int mini_batch_size
+		const char *split_prefix
+
+	int mm_set_opt(char *preset, mm_idxopt_t *io, mm_mapopt_t *mo)
+	int mm_verbose
+
+	#
+	# Indexing
+	#
+	ctypedef struct mm_idx_seq_t:
+		char *name
+		uint64_t offset
+		uint32_t len
+
+	ctypedef struct mm_idx_bucket_t:
+		pass
+
+	ctypedef struct mm_idx_t:
+		int32_t b, w, k, flag
+		uint32_t n_seq
+		mm_idx_seq_t *seq
+		uint32_t *S
+		mm_idx_bucket_t *B
+		void *km
+		void *h
+
+	ctypedef struct mm_idx_reader_t:
+		pass
+
+	mm_idx_reader_t *mm_idx_reader_open(const char *fn, const mm_idxopt_t *opt, const char *fn_out)
+	mm_idx_t *mm_idx_reader_read(mm_idx_reader_t *r, int n_threads)
+	void mm_idx_reader_close(mm_idx_reader_t *r)
+	void mm_idx_destroy(mm_idx_t *mi)
+	void mm_mapopt_update(mm_mapopt_t *opt, const mm_idx_t *mi)
+
+	int mm_idx_index_name(mm_idx_t *mi)
+
+	#
+	# Mapping (key struct defined in cmappy.h below)
+	#
+	ctypedef struct mm_reg1_t:
+		pass
+
+	ctypedef struct mm_tbuf_t:
+		pass
+
+	mm_tbuf_t *mm_tbuf_init()
+	void mm_tbuf_destroy(mm_tbuf_t *b)
+	void *mm_tbuf_get_km(mm_tbuf_t *b)
+	int mm_gen_cs(void *km, char **buf, int *max_len, const mm_idx_t *mi, const mm_reg1_t *r, const char *seq, int no_iden)
+	int mm_gen_MD(void *km, char **buf, int *max_len, const mm_idx_t *mi, const mm_reg1_t *r, const char *seq)
+
+#
+# Helper header (because it is hard to expose mm_reg1_t with Cython)
+#
+cdef extern from "cmappy.h":
+	ctypedef struct mm_hitpy_t:
+		const char *ctg
+		int32_t ctg_start, ctg_end
+		int32_t qry_start, qry_end
+		int32_t blen, mlen, NM, ctg_len
+		uint8_t mapq, is_primary
+		int8_t strand, trans_strand
+		int32_t seg_id
+		int32_t n_cigar32
+		uint32_t *cigar32
+
+	void mm_reg2hitpy(const mm_idx_t *mi, mm_reg1_t *r, mm_hitpy_t *h)
+	void mm_free_reg1(mm_reg1_t *r)
+	mm_reg1_t *mm_map_aux(const mm_idx_t *mi, const char *seq1, const char *seq2, int *n_regs, mm_tbuf_t *b, const mm_mapopt_t *opt)
+	char *mappy_fetch_seq(const mm_idx_t *mi, const char *name, int st, int en, int *l)
+	mm_idx_t *mappy_idx_seq(int w, int k, int is_hpc, int bucket_bits, const char *seq, int l)
+
+	ctypedef struct kstring_t:
+		unsigned l, m
+		char *s
+
+	ctypedef struct kstream_t:
+		pass
+
+	ctypedef struct kseq_t:
+		kstring_t name, comment, seq, qual
+		int last_char
+		kstream_t *f
+
+	kseq_t *mm_fastx_open(const char *fn)
+	void mm_fastx_close(kseq_t *ks)
+	int kseq_read(kseq_t *seq)
+
+	char *mappy_revcomp(int l, const uint8_t *seq)
+	int mm_verbose_level(int v)
+	void mm_reset_timer()

python/mappy.pyx

@@ -0,0 +1,251 @@
+from libc.stdint cimport uint8_t, int8_t
+from libc.stdlib cimport free
+cimport cmappy
+import sys
+
+__version__ = '2.13'
+
+cmappy.mm_reset_timer()
+
+cdef class Alignment:
+	cdef int _ctg_len, _r_st, _r_en
+	cdef int _q_st, _q_en
+	cdef int _NM, _mlen, _blen
+	cdef int8_t _strand, _trans_strand
+	cdef uint8_t _mapq, _is_primary
+	cdef int _seg_id
+	cdef _ctg, _cigar, _cs, _MD # these are python objects
+
+	def __cinit__(self, ctg, cl, cs, ce, strand, qs, qe, mapq, cigar, is_primary, mlen, blen, NM, trans_strand, seg_id, cs_str, MD_str):
+		self._ctg = ctg if isinstance(ctg, str) else ctg.decode()
+		self._ctg_len, self._r_st, self._r_en = cl, cs, ce
+		self._strand, self._q_st, self._q_en = strand, qs, qe
+		self._NM, self._mlen, self._blen = NM, mlen, blen
+		self._mapq = mapq
+		self._cigar = cigar
+		self._is_primary = is_primary
+		self._trans_strand = trans_strand
+		self._seg_id = seg_id
+		self._cs = cs_str
+		self._MD = MD_str
+
+	@property
+	def ctg(self): return self._ctg
+
+	@property
+	def ctg_len(self): return self._ctg_len
+
+	@property
+	def r_st(self): return self._r_st
+
+	@property
+	def r_en(self): return self._r_en
+
+	@property
+	def strand(self): return self._strand
+
+	@property
+	def trans_strand(self): return self._trans_strand
+
+	@property
+	def blen(self): return self._blen
+
+	@property
+	def mlen(self): return self._mlen
+
+	@property
+	def NM(self): return self._NM
+
+	@property
+	def is_primary(self): return (self._is_primary != 0)
+
+	@property
+	def q_st(self): return self._q_st
+
+	@property
+	def q_en(self): return self._q_en
+
+	@property
+	def mapq(self): return self._mapq
+
+	@property
+	def cigar(self): return self._cigar
+
+	@property
+	def read_num(self): return self._seg_id + 1
+
+	@property
+	def cs(self): return self._cs
+
+	@property
+	def MD(self): return self._MD
+
+	@property
+	def cigar_str(self):
+		return "".join(map(lambda x: str(x[0]) + 'MIDNSH'[x[1]], self._cigar))
+
+	def __str__(self):
+		if self._strand > 0: strand = '+'
+		elif self._strand < 0: strand = '-'
+		else: strand = '?'
+		if self._is_primary != 0: tp = 'tp:A:P'
+		else: tp = 'tp:A:S'
+		if self._trans_strand > 0: ts = 'ts:A:+'
+		elif self._trans_strand < 0: ts = 'ts:A:-'
+		else: ts = 'ts:A:.'
+		a = [str(self._q_st), str(self._q_en), strand, self._ctg, str(self._ctg_len), str(self._r_st), str(self._r_en),
+			str(self._mlen), str(self._blen), str(self._mapq), tp, ts, "cg:Z:" + self.cigar_str]
+		if self._cs != "": a.append("cs:Z:" + self._cs)
+		return "\t".join(a)
+
+cdef class ThreadBuffer:
+	cdef cmappy.mm_tbuf_t *_b
+
+	def __cinit__(self):
+		self._b = cmappy.mm_tbuf_init()
+
+	def __dealloc__(self):
+		cmappy.mm_tbuf_destroy(self._b)
+
+cdef class Aligner:
+	cdef cmappy.mm_idx_t *_idx
+	cdef cmappy.mm_idxopt_t idx_opt
+	cdef cmappy.mm_mapopt_t map_opt
+
+	def __cinit__(self, fn_idx_in=None, preset=None, k=None, w=None, min_cnt=None, min_chain_score=None, min_dp_score=None, bw=None, best_n=None, n_threads=3, fn_idx_out=None, max_frag_len=None, extra_flags=None, seq=None, scoring=None):
+		cmappy.mm_set_opt(NULL, &self.idx_opt, &self.map_opt) # set the default options
+		if preset is not None:
+			cmappy.mm_set_opt(str.encode(preset), &self.idx_opt, &self.map_opt) # apply preset
+		self.map_opt.flag |= 4 # always perform alignment
+		self.idx_opt.batch_size = 0x7fffffffffffffffL # always build a uni-part index
+		if k is not None: self.idx_opt.k = k
+		if w is not None: self.idx_opt.w = w
+		if min_cnt is not None: self.map_opt.min_cnt = min_cnt
+		if min_chain_score is not None: self.map_opt.min_chain_score = min_chain_score
+		if min_dp_score is not None: self.map_opt.min_dp_max = min_dp_score
+		if bw is not None: self.map_opt.bw = bw
+		if best_n is not None: self.map_opt.best_n = best_n
+		if max_frag_len is not None: self.map_opt.max_frag_len = max_frag_len
+		if extra_flags is not None: self.map_opt.flag |= extra_flags
+		if scoring is not None and len(scoring) >= 4:
+			self.map_opt.a, self.map_opt.b = scoring[0], scoring[1]
+			self.map_opt.q, self.map_opt.e = scoring[2], scoring[3]
+			self.map_opt.q2, self.map_opt.e2 = self.map_opt.q, self.map_opt.e
+			if len(scoring) >= 6:
+				self.map_opt.q2, self.map_opt.e2 = scoring[4], scoring[5]
+				if len(scoring) >= 7:
+					self.map_opt.sc_ambi = scoring[6]
+
+		cdef cmappy.mm_idx_reader_t *r;
+
+		if seq is None:
+			if fn_idx_out is None:
+				r = cmappy.mm_idx_reader_open(str.encode(fn_idx_in), &self.idx_opt, NULL)
+			else:
+				r = cmappy.mm_idx_reader_open(str.encode(fn_idx_in), &self.idx_opt, fn_idx_out)
+			if r is not NULL:
+				self._idx = cmappy.mm_idx_reader_read(r, n_threads) # NB: ONLY read the first part
+				cmappy.mm_idx_reader_close(r)
+				cmappy.mm_mapopt_update(&self.map_opt, self._idx)
+				cmappy.mm_idx_index_name(self._idx)
+		else:
+			self._idx = cmappy.mappy_idx_seq(self.idx_opt.w, self.idx_opt.k, self.idx_opt.flag&1, self.idx_opt.bucket_bits, str.encode(seq), len(seq))
+			cmappy.mm_mapopt_update(&self.map_opt, self._idx)
+			self.map_opt.mid_occ = 1000 # don't filter high-occ seeds
+
+	def __dealloc__(self):
+		if self._idx is not NULL:
+			cmappy.mm_idx_destroy(self._idx)
+
+	def __bool__(self):
+		return (self._idx != NULL)
+
+	def map(self, seq, seq2=None, buf=None, cs=False, MD=False, max_frag_len=None, extra_flags=None):
+		cdef cmappy.mm_reg1_t *regs
+		cdef cmappy.mm_hitpy_t h
+		cdef ThreadBuffer b
+		cdef int n_regs
+		cdef char *cs_str = NULL
+		cdef int l_cs_str, m_cs_str = 0
+		cdef void *km
+		cdef cmappy.mm_mapopt_t map_opt
+
+		map_opt = self.map_opt
+		if max_frag_len is not None: map_opt.max_frag_len = max_frag_len
+		if extra_flags is not None: map_opt.flag |= extra_flags
+
+		if self._idx is NULL: return None
+		if buf is None: b = ThreadBuffer()
+		else: b = buf
+		km = cmappy.mm_tbuf_get_km(b._b)
+
+		_seq = seq if isinstance(seq, bytes) else seq.encode()
+		if seq2 is None:
+			regs = cmappy.mm_map_aux(self._idx, _seq, NULL,  &n_regs, b._b, &map_opt)
+		else:
+			_seq2 = seq2 if isinstance(seq2, bytes) else seq2.encode()
+			regs = cmappy.mm_map_aux(self._idx, _seq, _seq2, &n_regs, b._b, &map_opt)
+
+		for i in range(n_regs):
+			cmappy.mm_reg2hitpy(self._idx, &regs[i], &h)
+			cigar, _cs, _MD = [], '', ''
+			for k in range(h.n_cigar32): # convert the 32-bit CIGAR encoding to Python array
+				c = h.cigar32[k]
+				cigar.append([c>>4, c&0xf])
+			if cs or MD: # generate the cs and/or the MD tag, if requested
+				if cs:
+					l_cs_str = cmappy.mm_gen_cs(km, &cs_str, &m_cs_str, self._idx, &regs[i], _seq, 1)
+					_cs = cs_str[:l_cs_str] if isinstance(cs_str, str) else cs_str[:l_cs_str].decode()
+				if MD:
+					l_cs_str = cmappy.mm_gen_MD(km, &cs_str, &m_cs_str, self._idx, &regs[i], _seq)
+					_MD = cs_str[:l_cs_str] if isinstance(cs_str, str) else cs_str[:l_cs_str].decode()
+			yield Alignment(h.ctg, h.ctg_len, h.ctg_start, h.ctg_end, h.strand, h.qry_start, h.qry_end, h.mapq, cigar, h.is_primary, h.mlen, h.blen, h.NM, h.trans_strand, h.seg_id, _cs, _MD)
+			cmappy.mm_free_reg1(&regs[i])
+		free(regs)
+		free(cs_str)
+
+	def seq(self, str name, int start=0, int end=0x7fffffff):
+		cdef int l
+		cdef char *s = cmappy.mappy_fetch_seq(self._idx, name.encode(), start, end, &l)
+		if l == 0: return None
+		r = s[:l] if isinstance(s, str) else s[:l].decode()
+		free(s)
+		return r
+
+	@property
+	def k(self): return self._idx.k
+
+	@property
+	def w(self): return self._idx.w
+
+	@property
+	def n_seq(self): return self._idx.n_seq
+
+def fastx_read(fn, read_comment=False):
+	cdef cmappy.kseq_t *ks
+	ks = cmappy.mm_fastx_open(str.encode(fn))
+	if ks is NULL: return None
+	while cmappy.kseq_read(ks) >= 0:
+		if ks.qual.l > 0: qual = ks.qual.s if isinstance(ks.qual.s, str) else ks.qual.s.decode()
+		else: qual = None
+		name = ks.name.s if isinstance(ks.name.s, str) else ks.name.s.decode()
+		seq = ks.seq.s if isinstance(ks.seq.s, str) else ks.seq.s.decode()
+		if read_comment:
+			if ks.comment.l > 0: comment = ks.comment.s if isinstance(ks.comment.s, str) else ks.comment.s.decode()
+			else: comment = None
+			yield name, seq, qual, comment
+		else:
+			yield name, seq, qual
+	cmappy.mm_fastx_close(ks)
+
+def revcomp(seq):
+	l = len(seq)
+	bseq = seq if isinstance(seq, bytes) else seq.encode()
+	cdef char *s = cmappy.mappy_revcomp(l, bseq)
+	r = s[:l] if isinstance(s, str) else s[:l].decode()
+	free(s)
+	return r
+
+def verbose(v=None):
+	if v is None: v = -1
+	return cmappy.mm_verbose_level(v)

python/minimap2.py

@@ -0,0 +1,39 @@
+#!/usr/bin/env python
+
+import sys
+import getopt
+import mappy as mp
+
+def main(argv):
+	opts, args = getopt.getopt(argv[1:], "x:n:m:k:w:r:c")
+	if len(args) < 2:
+		print("Usage: minimap2.py [options] <ref.fa>|<ref.mmi> <query.fq>")
+		print("Options:")
+		print("  -x STR      preset: sr, map-pb, map-ont, asm5, asm10 or splice")
+		print("  -n INT      mininum number of minimizers")
+		print("  -m INT      mininum chaining score")
+		print("  -k INT      k-mer length")
+		print("  -w INT      minimizer window length")
+		print("  -r INT      band width")
+		print("  -c          output the cs tag")
+		sys.exit(1)
+
+	preset = min_cnt = min_sc = k = w = bw = None
+	out_cs = False
+	for opt, arg in opts:
+		if opt == '-x': preset = arg
+		elif opt == '-n': min_cnt = int(arg)
+		elif opt == '-m': min_chain_score = int(arg)
+		elif opt == '-r': bw = int(arg)
+		elif opt == '-k': k = int(arg)
+		elif opt == '-w': w = int(arg)
+		elif opt == '-c': out_cs = True
+
+	a = mp.Aligner(args[0], preset=preset, min_cnt=min_cnt, min_chain_score=min_sc, k=k, w=w, bw=bw)
+	if not a: raise Exception("ERROR: failed to load/build index file '{}'".format(args[0]))
+	for name, seq, qual in mp.fastx_read(args[1]): # read one sequence
+		for h in a.map(seq, cs=out_cs): # traverse hits
+			print('{}\t{}\t{}'.format(name, len(seq), h))
+
+if __name__ == "__main__":
+	main(sys.argv)

sdust.c

@@ -56,6 +56,7 @@ sdust_buf_t *sdust_buf_init(void *km)
 	buf = (sdust_buf_t*)kcalloc(km, 1, sizeof(sdust_buf_t));
 	buf->km = km;
 	buf->w = kdq_init(int, buf->km);
+	kdq_resize(int, buf->w, 8);
 	return buf;
 }
 
@@ -69,10 +70,10 @@ void sdust_buf_destroy(sdust_buf_t *buf)
 static inline void shift_window(int t, kdq_t(int) *w, int T, int W, int *L, int *rw, int *rv, int *cw, int *cv)
 {
 	int s;
-	if (kdq_size(w) >= W - SD_WLEN + 1) { // TODO: is this right for SD_WLEN!=3?
+	if ((int)kdq_size(w) >= W - SD_WLEN + 1) { // TODO: is this right for SD_WLEN!=3?
 		s = *kdq_shift(int, w);
 		*rw -= --cw[s];
-		if (*L > kdq_size(w))
+		if (*L > (int)kdq_size(w))
 			--*L, *rv -= --cv[s];
 	}
 	kdq_push(int, w, t);
@@ -113,7 +114,7 @@ static void find_perfect(void *km, perf_intv_v *P, const kdq_t(int) *w, int T, i
 		r += c[t]++;
 		new_r = r, new_l = kdq_size(w) - i - 1;
 		if (new_r * 10 > T * new_l) {
-			for (j = 0; j < P->n && P->a[j].start >= i + start; ++j) { // find insertion position
+			for (j = 0; j < (int)P->n && P->a[j].start >= i + start; ++j) { // find insertion position
 				perf_intv_t *p = &P->a[j];
 				if (max_r == 0 || p->r * max_l > max_r * p->l)
 					max_r = p->r, max_l = p->l;
@@ -176,7 +177,7 @@ uint64_t *sdust(void *km, const uint8_t *seq, int l_seq, int T, int W, int *n)
 #ifdef _SDUST_MAIN
 #include <zlib.h>
 #include <stdio.h>
-#include <unistd.h>
+#include "ketopt.h"
 #include "kseq.h"
 KSEQ_INIT(gzFile, gzread)
 
@@ -185,16 +186,17 @@ int main(int argc, char *argv[])
 	gzFile fp;
 	kseq_t *ks;
 	int W = 64, T = 20, c;
+	ketopt_t o = KETOPT_INIT;
 
-	while ((c = getopt(argc, argv, "w:t:")) >= 0) {
-		if (c == 'w') W = atoi(optarg);
-		else if (c == 't') T = atoi(optarg);
+	while ((c = ketopt(&o, argc, argv, 1, "w:t:", 0)) >= 0) {
+		if (c == 'w') W = atoi(o.arg);
+		else if (c == 't') T = atoi(o.arg);
 	}
-	if (optind == argc) {
+	if (o.ind == argc) {
 		fprintf(stderr, "Usage: sdust [-w %d] [-t %d] <in.fa>\n", W, T);
 		return 1;
 	}
-	fp = strcmp(argv[optind], "-")? gzopen(argv[optind], "r") : gzdopen(fileno(stdin), "r");
+	fp = strcmp(argv[o.ind], "-")? gzopen(argv[o.ind], "r") : gzdopen(fileno(stdin), "r");
 	ks = kseq_init(fp);
 	while (kseq_read(ks) >= 0) {
 		uint64_t *r;

setup.py

@@ -0,0 +1,65 @@
+try:
+	from setuptools import setup, Extension
+except ImportError:
+	from distutils.core import setup
+	from distutils.extension import Extension
+
+cmdclass = {}
+
+try:
+	from Cython.Build import build_ext
+except ImportError: # without Cython
+	module_src = 'python/mappy.c'
+else: # with Cython
+	module_src = 'python/mappy.pyx'
+	cmdclass['build_ext'] = build_ext
+
+import sys, platform
+
+sys.path.append('python')
+
+extra_compile_args = ['-DHAVE_KALLOC']
+include_dirs = ["."]
+
+if platform.machine() in ["aarch64", "arm64"]:
+	include_dirs.append("sse2neon/")
+	extra_compile_args.extend(['-ftree-vectorize', '-DKSW_SSE2_ONLY', '-D__SSE2__'])
+else:
+	extra_compile_args.append('-msse4.1') # WARNING: ancient x86_64 CPUs don't have SSE4
+
+def readme():
+	with open('python/README.rst') as f:
+		return f.read()
+
+setup(
+	name = 'mappy',
+	version = '2.13',
+	url = 'https://github.com/lh3/minimap2',
+	description = 'Minimap2 python binding',
+	long_description = readme(),
+	author = 'Heng Li',
+	author_email = 'lh3@me.com',
+	license = 'MIT',
+	keywords = 'sequence-alignment',
+	scripts = ['python/minimap2.py'],
+    ext_modules = [Extension('mappy',
+		sources = [module_src, 'align.c', 'bseq.c', 'chain.c', 'format.c', 'hit.c', 'index.c', 'pe.c', 'options.c',
+				   'ksw2_extd2_sse.c', 'ksw2_exts2_sse.c', 'ksw2_extz2_sse.c', 'ksw2_ll_sse.c',
+				   'kalloc.c', 'kthread.c', 'map.c', 'misc.c', 'sdust.c', 'sketch.c', 'esterr.c', 'splitidx.c'],
+		depends = ['minimap.h', 'bseq.h', 'kalloc.h', 'kdq.h', 'khash.h', 'kseq.h', 'ksort.h',
+				   'ksw2.h', 'kthread.h', 'kvec.h', 'mmpriv.h', 'sdust.h',
+				   'python/cmappy.h', 'python/cmappy.pxd'],
+		extra_compile_args = extra_compile_args,
+		include_dirs = include_dirs,
+		libraries = ['z', 'm', 'pthread'])],
+	classifiers = [
+		'Development Status :: 5 - Production/Stable',
+		'License :: OSI Approved :: MIT License',
+		'Operating System :: POSIX',
+		'Programming Language :: C',
+		'Programming Language :: Cython',
+		'Programming Language :: Python :: 2.7',
+		'Programming Language :: Python :: 3',
+		'Intended Audience :: Science/Research',
+		'Topic :: Scientific/Engineering :: Bio-Informatics'],
+	cmdclass = cmdclass)

sketch.c

@@ -2,25 +2,26 @@
 #include <stdlib.h>
 #include <assert.h>
 #include <string.h>
+#define __STDC_LIMIT_MACROS
 #include "kvec.h"
-#include "minimap.h"
+#include "mmpriv.h"
 
 unsigned char seq_nt4_table[256] = {
-	0, 1, 2, 3,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
+	0, 1, 2, 3,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 0, 4, 1,  4, 4, 4, 2,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  3, 3, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 0, 4, 1,  4, 4, 4, 2,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  3, 3, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
+	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
 	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 0, 4, 1,  4, 4, 4, 2,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  3, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 0, 4, 1,  4, 4, 4, 2,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  3, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
-	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4, 
 	4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4,  4, 4, 4, 4
 };
 
@@ -77,11 +78,10 @@ void mm_sketch(void *km, const char *str, int len, int w, int k, uint32_t rid, i
 {
 	uint64_t shift1 = 2 * (k - 1), mask = (1ULL<<2*k) - 1, kmer[2] = {0,0};
 	int i, j, l, buf_pos, min_pos, kmer_span = 0;
-	mm128_t *buf, min = { UINT64_MAX, UINT64_MAX };
+	mm128_t buf[256], min = { UINT64_MAX, UINT64_MAX };
 	tiny_queue_t tq;
 
-	assert(len > 0 && w > 0 && k > 0 && k <= 28); // 56 bits for k-mer; could use long k-mers, but 28 enough in practice
-	buf = (mm128_t*)alloca(w * 16);
+	assert(len > 0 && (w > 0 && w < 256) && (k > 0 && k <= 28)); // 56 bits for k-mer; could use long k-mers, but 28 enough in practice
 	memset(buf, 0xff, w * 16);
 	memset(&tq, 0, sizeof(tiny_queue_t));
 	kv_resize(mm128_t, km, *p, p->n + len/w);
@@ -102,34 +102,34 @@ void mm_sketch(void *km, const char *str, int len, int w, int k, uint32_t rid, i
 				tq_push(&tq, skip_len);
 				kmer_span += skip_len;
 				if (tq.count > k) kmer_span -= tq_shift(&tq);
-				if (kmer_span >= 256) continue; // make sure $kmer_span does not take more than 8 bits
 			} else kmer_span = l + 1 < k? l + 1 : k;
 			kmer[0] = (kmer[0] << 2 | c) & mask;           // forward k-mer
 			kmer[1] = (kmer[1] >> 2) | (3ULL^c) << shift1; // reverse k-mer
 			if (kmer[0] == kmer[1]) continue; // skip "symmetric k-mers" as we don't know it strand
 			z = kmer[0] < kmer[1]? 0 : 1; // strand
-			if (++l >= k) {
+			++l;
+			if (l >= k && kmer_span < 256) {
 				info.x = hash64(kmer[z], mask) << 8 | kmer_span;
 				info.y = (uint64_t)rid<<32 | (uint32_t)i<<1 | z;
 			}
 		} else l = 0, tq.count = tq.front = 0, kmer_span = 0;
 		buf[buf_pos] = info; // need to do this here as appropriate buf_pos and buf[buf_pos] are needed below
-		if (l == w + k - 1) { // special case for the first window - because identical k-mers are not stored yet
+		if (l == w + k - 1 && min.x != UINT64_MAX) { // special case for the first window - because identical k-mers are not stored yet
 			for (j = buf_pos + 1; j < w; ++j)
 				if (min.x == buf[j].x && buf[j].y != min.y) kv_push(mm128_t, km, *p, buf[j]);
 			for (j = 0; j < buf_pos; ++j)
 				if (min.x == buf[j].x && buf[j].y != min.y) kv_push(mm128_t, km, *p, buf[j]);
 		}
 		if (info.x <= min.x) { // a new minimum; then write the old min
-			if (l >= w + k) kv_push(mm128_t, km, *p, min);
+			if (l >= w + k && min.x != UINT64_MAX) kv_push(mm128_t, km, *p, min);
 			min = info, min_pos = buf_pos;
 		} else if (buf_pos == min_pos) { // old min has moved outside the window
-			if (l >= w + k - 1) kv_push(mm128_t, km, *p, min);
+			if (l >= w + k - 1 && min.x != UINT64_MAX) kv_push(mm128_t, km, *p, min);
 			for (j = buf_pos + 1, min.x = UINT64_MAX; j < w; ++j) // the two loops are necessary when there are identical k-mers
 				if (min.x >= buf[j].x) min = buf[j], min_pos = j; // >= is important s.t. min is always the closest k-mer
 			for (j = 0; j <= buf_pos; ++j)
 				if (min.x >= buf[j].x) min = buf[j], min_pos = j;
-			if (l >= w + k - 1) { // write identical k-mers
+			if (l >= w + k - 1 && min.x != UINT64_MAX) { // write identical k-mers
 				for (j = buf_pos + 1; j < w; ++j) // these two loops make sure the output is sorted
 					if (min.x == buf[j].x && min.y != buf[j].y) kv_push(mm128_t, km, *p, buf[j]);
 				for (j = 0; j <= buf_pos; ++j)

splitidx.c

@@ -0,0 +1,80 @@
+#include <string.h>
+#include <assert.h>
+#include <stdlib.h>
+#include <stdio.h>
+#include "mmpriv.h"
+
+FILE *mm_split_init(const char *prefix, const mm_idx_t *mi)
+{
+	char *fn;
+	FILE *fp;
+	uint32_t i, k = mi->k;
+	fn = (char*)calloc(strlen(prefix) + 10, 1);
+	sprintf(fn, "%s.%.4d.tmp", prefix, mi->index);
+	fp = fopen(fn, "wb");
+	assert(fp);
+	mm_err_fwrite(&k, 4, 1, fp);
+	mm_err_fwrite(&mi->n_seq, 4, 1, fp);
+	for (i = 0; i < mi->n_seq; ++i) {
+		uint8_t l;
+		l = strlen(mi->seq[i].name);
+		mm_err_fwrite(&l, 1, 1, fp);
+		mm_err_fwrite(mi->seq[i].name, 1, l, fp);
+		mm_err_fwrite(&mi->seq[i].len, 4, 1, fp);
+	}
+	free(fn);
+	return fp;
+}
+
+mm_idx_t *mm_split_merge_prep(const char *prefix, int n_splits, FILE **fp, uint32_t *n_seq_part)
+{
+	mm_idx_t *mi = 0;
+	char *fn;
+	int i, j;
+
+	if (n_splits < 1) return 0;
+	fn = CALLOC(char, strlen(prefix) + 10);
+	for (i = 0; i < n_splits; ++i) {
+		sprintf(fn, "%s.%.4d.tmp", prefix, i);
+		if ((fp[i] = fopen(fn, "rb")) == 0) {
+			if (mm_verbose >= 1)
+				fprintf(stderr, "ERROR: failed to open temporary file '%s'\n", fn);
+			for (j = 0; j < i; ++j)
+				fclose(fp[j]);
+			free(fn);
+			return 0;
+		}
+	}
+	free(fn);
+
+	mi = CALLOC(mm_idx_t, 1);
+	for (i = 0; i < n_splits; ++i) {
+		mm_err_fread(&mi->k, 4, 1, fp[i]); // TODO: check if k is all the same
+		mm_err_fread(&n_seq_part[i], 4, 1, fp[i]);
+		mi->n_seq += n_seq_part[i];
+	}
+	mi->seq = CALLOC(mm_idx_seq_t, mi->n_seq);
+	for (i = j = 0; i < n_splits; ++i) {
+		uint32_t k;
+		for (k = 0; k < n_seq_part[i]; ++k, ++j) {
+			uint8_t l;
+			mm_err_fread(&l, 1, 1, fp[i]);
+			mi->seq[j].name = (char*)calloc(l + 1, 1);
+			mm_err_fread(mi->seq[j].name, 1, l, fp[i]);
+			mm_err_fread(&mi->seq[j].len, 4, 1, fp[i]);
+		}
+	}
+	return mi;
+}
+
+void mm_split_rm_tmp(const char *prefix, int n_splits)
+{
+	int i;
+	char *fn;
+	fn = CALLOC(char, strlen(prefix) + 10);
+	for (i = 0; i < n_splits; ++i) {
+		sprintf(fn, "%s.%.4d.tmp", prefix, i);
+		remove(fn);
+	}
+	free(fn);
+}

test/MT-orang.fa

@@ -1,4 +1,4 @@
->MT_orang
+>MT_orang co:Z:comment
 GTTTATGTAGCTTATTCTATCCAAAGCAATGCACTGAAAATGTCTCGACGGGCCCACACG
 CCCCATAAACAAATAGGTTTGGTCCTAGCCTTTCTATTAGCTCTTAGTGAGGTTACACAT
 GCAAGCATCCCCGCCCCAGTGAGTCGCCCTCCAAGTCACTCTGACTAAGAGGAGCAAGCA

test/t-inv.fa

@@ -0,0 +1,127 @@
+>ref
+TGCGGAGGCTGAAGCAACTCCATCTTGGAAGCTAATCTACCATGTTGGCTTCTGATTAAC
+ATCAGTTCTGGGAAGGCTTGTAAGATTTCCTGTTTGTCTATTATTTCCTAGGTAAGAGCA
+GATACTTACTGTAAATCCTGCCCCTAGATTAAACAACCTTGGTGTTATCGTACTTCCATT
+GTCCTATACATCCCTTCGGAATCCCCCTTTCCCTATGGTCCTCAAGCCCTTGGTCTGGGG
+AGTAACAGCATAGGGATCAACCATCTCGTCTTGCCACTGCCCGAAATACAGACATGGCTT
+CTGTTCCTAAGTCCCTATTCAACTTTTCTTTCTAAGAAACTGGATTTGTCAGCCTCTTTC
+TTCACCTCTCAGCTTCCTTGGACTTTGGGGGTAGGTTTGCGTAGACATGCTCACCACAGA
+CACAATATCAGCTTCATTCTACAGATGAGGAAGGCAAGCCTTGGGGAGCTTAACCAACTT
+GTCGAGACTCATGTATATACCAACACTGAAAAGCAGATATTCCAGACTCCCAGTCATGCC
+ACAGGCACACCCCTCAGTGAGAGGTGGGGTTTGTAGTTGAGGCTATTTCCTGCCCAGGGA
+GCAGGGAGGCACTCTAGCTTCCCTGAGCTAACGTGGTTCTGCTTGTGTCTGACTTCCAGG
+TCTCTGCCCTTTCCAAGCTCACTAGGATGGGCTTCGGGTGTGTCAAATGCCTCAGACAGT
+ACAGATCCACACAGAATGGGCATATGCAACCAATCAGTGTCATAAAAAAGAAGGAAATGA
+CTCGGGCCCCCTGTGTGTTCAACATGTCGAAGGTATCTGTGCAGCAGAAGAAAGAGGGGC
+AAAAGCCCCCAGTGCCACAGGCCAGAGGCAGCAGCTTGGGCCCATGTGGGAGGGTTTGCT
+TTCCCCTGCCAAAGTGATGGGCTGCTGCAGCCTGGGGCTTGTGGGAATCCTTCCTGGGCC
+TGTGTGGGAAGTGTAGGCAGGGAGAGTGCTGCTTTCCCAAGCTCATCCCAGCTACAGCTA
+CCTTTGTGCTCTGGGATTCAGGACCCCCGAGGGGGCTGGCAGGAGAGTCTCTGTTCTCGG
+ATGGGTTGTCACCAGGGCATACATGGGAAGTGGGCTCTCTGGAGTCACCCTCCAGGGGAC
+AATGCCAATTCCAGACACATTTACTGGAACCCCTACACTGATGACCTTTTGTTGAGGGTT
+GAATTATGTCCCCAAAAAAGATACATTGAAGTCCAAACCTCTGGTGTCTATAAATGTGAT
+TTTATTTGAAAATGAGGTTTCTATGGACTAAATTGTGTCCCTCCCAAATTCATATTTTGA
+AGCCCTAGCCCCCAGTGTGACTATACCTAGAGACAGAGATCTTTAGGAGGTAATTAAGGT
+TCAATGAGGTCAGGTGGGTGGGGCCCTAAACCAACAGGAAGGACTGTGGCCTTACTAGAA
+AAGGAAGAAAAAGCATTTCCTCTCTTCTAGTATAAAAGGACACAGAAAGAAGGCAGATAT
+CTACAAGCCACGAAGAGAGACGTCACTGAGAACTGAATTTGTGTACATTGATCTGGAACT
+TCCAGCCTCCAGAACTTGAGAAATACATTTCTGTTGTTTATTTTTTTTTCATGTAATCAA
+TTCATTTATCATATATTTATTGAGTGCCTACTATGTGCCAGAGGATACAGCAGTAACAAA
+ACTAGGCAAAAATTGTGCCTAAAAGAGGGAAGATGACTTTTCTTAAAGTGTGGAATAAAG
+AAAAGTAAGATAGCGGATAGAAGCTTGAAGTGAAAGCAGGTTCACAGGAAGTTTCTTTGG
+TCATTTGTTTTGTTTTTAAATAGTGGAAAGATGTATATGTTTATGGAGAAAGATTGCCTT
+GAAGATGCAAGAGGAAGAGATGATCAAAATTCAAGAAGAAGCAGAAAGTGATAGAATAAA
+GAGCACAAGTGGAGAATTAGTGTTAATGAAAAGAAGGATGCTTCCTTTGATATGAAGTGA
+AGGAAGAGAGAATGAGTAAAGACCAAGACTTGAAGTCCCTAGTTTAATAGAGGGAGATTT
+CTTCTTTTGATAGCAACAATGGTATTCTGAATTATTTGAAGACATGTCATATTTCTCTTG
+TGCCATTTTCCTCCCAGTTTAAACATTCTCATAACCTCTATTCCTCACATGATGTTTTTC
+CAGGTCCTTTATTCTTTGGCACTCTCTTCTCTGGACACATTGTATTCTGTCATTGGTCCT
+AAAATTTAGATACCCACAATTGAACATACTCCTCTAGATATGGTCTAGCTAATGCAAAAG
+AACTGCTGCCTTCCAACTTGTTCAGACATCATATGTTTGTTGTCAAACGCTAAGTTGAGT
+TGTTATCTTTTAAGTTTTGTTTTTGTTTTTTTTTTTTTTTTTTAATTCCAAGAGGTGCCC
+ACGTTGGCTAAGTACCAAACAGGGTACTAGGGAATTTTACTTCTGAGTTAAATGCCATTC
+TAGTTGTTTTTTCTTCATCTCCAGTAAGGTTATCTTTATTCACCAGTTGTTACAATAGCT
+GTGGGTCTTGCTTCTCACAGTTTTATGCTGTCTGTGCTATTTTCTCTACTGATCATCACC
+ACAATCATTATTGCTTATCATAATTGTTATCTTTATTTTCTCCTTTAATCAAGAATCAGT
+CTTCCTTTATCTCATTATTCTCTTTTGCAGGCTTCAGGATAATTATGGTTGGAGTGCACT
+GGGGGAACCAGTGCAGCTAAGCTCTGACATCTTTGCATCCCTTTTCCATCTGCTGTTTTG
+GCACTCTGGTAGAATAGATAACCTAAAAACGACTTTAAAACATCTAGAAATTTTGGATAA
+AATATAACAAACATCCCTTTAAATGCACAACTGATCTTCCATGGAAGTCACAGAAATATA
+TAACGCCAAAAAGAAGGGAAGCTGAAACCCAGGGCTGTAAACATGAACATCATCTTCTCT
+CCCTTTTTCTTGTGACTTATCTTGTTTTTCTCAGCTTTGGTGCTACCAAGGCTTGACTTT
+AATAGGCATTTCCAATCAATGAGAGAATTTCTTTTGCTTTCATCAACAATTCAGTTATTG
+ATGTTAACATATATATCATTTGAGTACTTTTCTTTTTTTTATTATTATTATACTTTAAGT
+TTTAGGGTCCATGTGCACAATGTGCAGGTTAGTTACGTATGTATACATGTGCCATGCTGG
+TGTGCTGCACCCATTAACTCATCATTTAGCATTAGGTATATCTCCTAATGCTATCCCTTC
+CCCCTCTCCCCACCCCACAACAGTCCCCAGAGTGTTCCCCTTCCTGTGTCCATGTGTTCT
+CATTGTTCAATCCCCATCTATGAGTGAGAACATGCGGTGTTTGGTTTTTTGTCCTTGCAA
+TAGTTTACTGAGAATGATGATTTCTAATTTCATCCATGTCCCTAAAGAGCTTCTGCACAG
+CAAAAGAAACTACCATCAGAGTGAACAGGCAACCTACAAAATGGGAGAAAATTTTCACAA
+CCTGCTCATCTGACAAAGGGCTAATATCCAGAATCTACAATGAACTCAAACAAATTTACA
+AGAAAAAAACAAACAACCCCATCAAAAAGTGGGCAAAGGATATGAACAGACACTTCTCAA
+AAGAAGACATTTATGCAGCCAAAAGACACATGAAAAAATGCTCATCATCACTGGCCATCA
+GAGAAATGCAAACCAAAACCACAATGAGATACCATCTCACACCAGTTAAAATGGCAATCA
+TTAAAAAGTCAGGAAACAACAGGTGCTGGAGAGGATGTGGAGAAACAGGAACACTTTTAC
+ACTGTTGGTGGGACTGTAAACTAGTTCAACCATTGTGGAAGTCAGTGTGCTGATTCCTCA
+GGGATCTAGAACTAGAAATACCATTTGACCCAGCCATCCCATTACTGGGTATATACCCAA
+AGGACTATAAATCATGCTGCTATAAAGACACATGCACACGTATGTTTATTGCGGCACTAT
+TCACAATAGCAAAGACTTGGAACCAACCCAAATGTCCAACAATGATAGACTGGATTAAGA
+AAATGTGGCACATATACACCACGGAATACTGTGCAGCCATAAAAAATGATGAGTTCATGT
+CCTTTGTAGGGACACGGATGAAATTGGAAATCATTTCTGTTGTTTAAACCACGAAGTCTA
+TGGTATCTGGTTATGACAACCTGAGAATACTAACTCAAGGGTCTTTCGCAGATGTCATTA
+AGTTGTTAAAGTGAGGTCATTATGGTGGGTCCTAATCCAAGAGAAGAGATGCATGGACAG
+ACGTGCACAACGGGAGGACCAAGCCAAGACACACAGGGAGAATGGCCATGGGAAGATGGA
+GGCAGAGATCAAAGTGAGGCACCCACAAGCCAAGAAATGGCAGGAGCTACCAGCAGCTGG
+AAGATGCAGAGAAGCATTCCTTCTTAGAGGTTTCAGAGAGAGTATGGTGCTACTGACACC
+TTGATTTTGAACTTCTAGTCTCCAGAACTATGAGAGAATAAATTTCTGTTGGTTAAGCCA
+TCGAGTTTGTGTAAGTTTGTTATAAGAGCCCTAGGAAATAAACATATCCATTTATTCAGG
+AAAGCCTGCTAGAGTGCAAATATTTGGAAAAGATACTACTATGCAAATGTTTGAAAAAGA
+TATTGCTCTTGATTCTGCCTTATGGGTTTTTCATTTCTGTAAGCTATTCTCAAAGTTTTG
+TTCTTGGACTACTATTGGTAATTAAGACTGCAACATGTTTGGCAACATCAGTTGAGAACT
+GTTGCTCTGGGAACGTTTTCGGCAAGCCTCAGCCCTTCTTTTCCCTTGGCTTGCATTGAG
+GAGTTAGGTGATACTCTGCTGCTCAGGCCCAGCACCTTTATGGACCGTATTCCCCTGGTG
+GAATGACCATCTCTGCTTGCTCTGATTGGCTGTTGGGGTTTTCTAGCATGCCCTATTTAA
+TATGTATGATTTATCTCTTACTTCAGTTGGAAGGTACAGTTGCTCTGTAGTTGGCATGCA
+GTCATGGTGACTATGAAAATATAAAATAATGTTTTGGTTTACAGACACTTAGAAATAAGT
+TGTGTCTCAAAATTGGGTGACTATTCTAGTTATCTGCTACTCAATATCCTTGTGCGAGCC
+CTCTTTACCCAGAATCAAACTAAACCATGAGGGGCACTATAGAATGTCACCCCTGGGTCC
+AGGATACTATGGGGACTCAGAAGCCAAGCTCCCACTGGGGGATCTAGGGCATGCCCCCAA
+GGTAAGATTCCCACCTCTTTGTTCAGCAGGAAGCACCCATCACACAAGGAGGTAGGAATA
+AACAAGCATTCGTCAAGAACAAAAGATACAGATGTTCTGCTGGAGCTTGGATACATAGCA
+TAAGAGGGAACAGTTCTCACAGGTAAGAGTAAGTTTTCCTCTGGTGGTGACAGTGGGACC
+TGTGGGGGAGAGAATTGGGAGTACTGACAGGAAGGCAGAGTGGCTGTCCAAATGAACGGA
+TTGTTTGCACATGGCCTTTAGGGCACGTTGTGTTAGCCTTCCATTGCTGCTTATATTAGT
+CTGTTTTCACACTGCCCATAAATGCATACCTGAGACTGGATAATTTATAAAGAAAAAGAG
+CCTTAATGTACTCATAGTTGCATGTGGCTGGGGAGGCCTCACAATCATGGCAGAAGGTGA
+AAGGCACATCTTACATGGAAGCAGACAAGAGAGAATTGAGGACCAAGTGAAAGGGGTTTC
+CCCTTATAAAACCATCAGATCACATGAGACTTTTTCACCACCATGAGAACAGTAAGGGGA
+AAACTATGCTCATGATTCAATTGTCTCCCACTGGATTCCTCCCACAACACATAGGAATTA
+TGGGAGCTAAAATTCAAGATGAGATTTGGGTGAGGACACAGCCAAACCCTATCACTGCTG
+TAATCAATTCCCACCAACTTAGTGGCTCGAAACATCACAGATTTATGATCTTATGACGGT
+GGAGGTCCCCAAATGGATCTTCTAGGTCTAGAATCAAGGTATCAGCAGACCACTTCTTTT
+GGAGGCTCTGGTGGAGAAACCATTTCCTCGCCTTTTCCAGCTTCTAGAGGCTGCCCTTCT
+CATTCCTTGGTTCACGGCCACACTCATTTCCATCTCTGCTTCCACTGTGACAACTTCTCT
+GCCTCAGACCCTCCTGCTTTGCCTTTGTAAGGACCCTTGTGATGAGATCAGGCCCATCCA
+GGATTATCCCTCATCTCAAGACCTTTACCTTAATCACATTTGCAAGGTCTCTTCCACTGT
+GTCAGGTAACATTTTCACAGGTTCCAGGGATTAGGGTGTGGACATCTTGGGGAGCTGGAG
+GATATTATTTCATCTACCACACACATCTCTACCTTGTACAGGCAAGCACTTGCAAAGTGC
+AATGTGATCCTCTGGAGCCACTGTCCTCCCAGAGCTTATATATACTCTGAAAGTCAACTC
+TCAGACCACAGCCTCCTGTCCATGCACCACTCTCATCAACACCCCCACCCGAAACACTTT
+CACTCCACCCTCTTTGTCCCCTAACTCATGGAGAAGAAAATCTAATTAGTAGGAGTGGAA
+TTTGGCTTTCATCTTTACCAGTACTAGAAATATGGTGTGTGTCTTTTTGTAAAAATTCTC
+TCAACTAAATTGTTTTTATTAATTTCTGCAAAATGTGAACATCAACTCCCTTCATGTGAA
+TGTCAATAAGATTAAATGAGCTGTCTCAGCTCCTAGCCTGTGCAAGCTAACAGCTCAGGA
+GATGTTTATTTCTTTCCCTCTTCTTTCCTTAATGAAGCCCTCTCCTTTGACATCTTCAAT
+TCTGGAGCGCTTCTTTTCTGAGGCCTTGGCTCCCCCACATTGCCCACCCTTTTCCTGCTC
+GTCCACATTTCTGGCTTCTATTCTCTTGTCTTTACCATCTCCCTGAACAATGTTATCCGT
+TCCAATGACTTCAACAGTCTCTCCGCTTACATATGATGCCTCTCAAACTCTGATCTCCAA
+CTCTTCCAAAGAGCTCTGGACCTTTGTTCCAATTACCTGAAAAACATCTTCTTGGATGTC
+CCATTAGCACTGTTAAATCAAACAAGAATTTCCCTCCCTCCTGCCTTGCTGTAGTTCCCC
+TAGGGATTCGGTTGTGTGGGAAGATGTGTGGAGAGCTCTTAGTTGACTCCCTTCTCTGCA
+GTTCTACCTCTCTAGAGACTTGGAGGACCCACTGTTTCCGCCTCGCTTTTTCAGGCCTAG
+AGATTGCTCGCTCCTGGGCTGGCTGCTTCATAATTCCTTATTAGTAGTTTCCCAAGCTTA
+CATATCTGTAAATATTTACTTTAGTTAAATTCTCCCCAATTTCCACAATATGTTGGCTGC
+ACATGCTTTCTACTAGGAGTCACACAACTATGATAAGAACCAAGAAATATTAGTAAACGT
+TTTTTACCATTATTGGCCTATACCCTGGAATAGCCAACAATAACCTAGAACCTATGCAAC
+AAGAATATCCAACAAGAACCTAGAGACCTGTCAGTCTATAGGTGGGAACTACAGGATGAG
+A

tex/bowtie2-s3.sam.eval

@@ -0,0 +1,28 @@
+Q	42	16872292	669	0.000039651	16872292
+Q	40	835329	636	0.000073697	17707621
+Q	31	6544	2	0.000073783	17714165
+Q	30	8882	6	0.000074084	17723047
+Q	27	68499	9	0.000074305	17791546
+Q	26	132041	81	0.000078277	17923587
+Q	25	129378	96	0.000083033	18052965
+Q	24	92056	382	0.000103665	18145021
+Q	23	14341	402	0.000125720	18159362
+Q	22	132838	146	0.000132789	18292200
+Q	21	122274	124	0.000138641	18414474
+Q	18	112183	103	0.000143361	18526657
+Q	17	126981	213	0.000153804	18653638
+Q	16	16356	208	0.000164810	18669994
+Q	15	42804	782	0.000206223	18712798
+Q	14	16026	318	0.000223025	18728824
+Q	12	170250	814	0.000264087	18899074
+Q	11	48351	1409	0.000337777	18947425
+Q	8	1843	311	0.000354156	18949268
+Q	7	62266	4435	0.000586276	19011534
+Q	6	413997	50057	0.003150647	19425531
+Q	5	404	58	0.003153568	19425935
+Q	4	704	154	0.003161381	19426639
+Q	3	1473	681	0.003196193	19428112
+Q	2	17541	16462	0.004039875	19445653
+Q	1	534344	354879	0.021693547	19979997
+Q	0	11939	9917	0.022176642	19991936
+U	8064

tex/bwa-s3.sam.eval

@@ -0,0 +1,52 @@
+Q	60	18784147	3	0.000000160	18784147
+Q	52	19002	1	0.000000213	18803149
+Q	50	7152	2	0.000000319	18810301
+Q	49	6797	1	0.000000372	18817098
+Q	48	52188	2	0.000000477	18869286
+Q	47	48775	3	0.000000634	18918061
+Q	46	19447	2	0.000000739	18937508
+Q	45	25983	3	0.000000896	18963491
+Q	44	13455	1	0.000000949	18976946
+Q	43	14573	2	0.000001053	18991519
+Q	42	8697	4	0.000001263	19000216
+Q	41	8645	2	0.000001368	19008861
+Q	40	176603	75	0.000005264	19185464
+Q	38	2503	2	0.000005368	19187967
+Q	37	4117	3	0.000005523	19192084
+Q	36	2924	16	0.000006356	19195008
+Q	35	2323	8	0.000006772	19197331
+Q	34	2344	10	0.000007292	19199675
+Q	33	4279	6	0.000007603	19203954
+Q	32	2092	4	0.000007810	19206046
+Q	31	2625	11	0.000008382	19208671
+Q	30	2828	13	0.000009057	19211499
+Q	29	1581	1	0.000009108	19213080
+Q	28	1543	6	0.000009420	19214623
+Q	27	70916	223	0.000020948	19285539
+Q	26	1288	16	0.000021777	19286827
+Q	25	25551	122	0.000028065	19312378
+Q	24	14345	84	0.000032390	19326723
+Q	23	7308	87	0.000036878	19334031
+Q	22	8358	125	0.000043325	19342389
+Q	21	4836	71	0.000046983	19347225
+Q	20	5888	123	0.000053325	19353113
+Q	19	4656	83	0.000057600	19357769
+Q	18	3948	87	0.000062081	19361717
+Q	17	4418	114	0.000067954	19366135
+Q	16	4226	131	0.000074702	19370361
+Q	15	5760	164	0.000083144	19376121
+Q	14	4697	257	0.000096384	19380818
+Q	13	5246	313	0.000112503	19386064
+Q	12	4170	241	0.000124908	19390234
+Q	11	4095	304	0.000140557	19394329
+Q	10	3857	360	0.000159087	19398186
+Q	9	5300	438	0.000181617	19403486
+Q	8	4206	572	0.000211050	19407692
+Q	7	4676	787	0.000251541	19412368
+Q	6	3923	688	0.000286924	19416291
+Q	5	3294	708	0.000323333	19419585
+Q	4	2936	693	0.000358965	19422521
+Q	3	3928	816	0.000400897	19426449
+Q	2	2613	810	0.000442533	19429062
+Q	1	3515	1188	0.000503587	19432577
+Q	0	567423	376636	0.019321100	20000000

tex/hs38-simu.sh

@@ -2,3 +2,9 @@
 
 bin/mason_variator -ir hs38.fa -s 1 -ov hs38-s1.vcf --snp-rate 1e-3 --small-indel-rate 2e-4 --sv-indel-rate 0 --sv-inversion-rate 0 --sv-translocation-rate 0 --sv-duplication-rate 0 --max-small-indel-size 10
 bin/mason_simulator -ir hs38.fa -iv hs38-s1.vcf -n 1000000 --seed 1 -o s1_1.fq -or s1_2.fq -oa s1.sam --illumina-prob-mismatch-scale 2.5
+
+bin/mason_variator -ir hs38.fa -s 2 -ov hs38-s2.vcf --snp-rate 1e-3 --small-indel-rate 2e-4 --sv-indel-rate 0 --sv-inversion-rate 0 --sv-translocation-rate 0 --sv-duplication-rate 0 --max-small-indel-size 10
+bin/mason_simulator -ir hs38.fa -iv hs38-s2.vcf -n 1000000 --seed 2 -o mason-s2_1.fq -or mason-s2_2.fq -oa mason-s2.sam --illumina-prob-mismatch-scale 2.5 --illumina-read-length 150
+
+bin/mason_variator -ir hs38.fa -s 3 -ov hs38-s3.vcf --snp-rate 1e-3 --small-indel-rate 2e-4 --sv-indel-rate 0 --sv-inversion-rate 0 --sv-translocation-rate 0 --sv-duplication-rate 0 --max-small-indel-size 10
+bin/mason_simulator -ir hs38.fa -iv hs38-s3.vcf -n 10000000 --seed 3 -o mason-s3_1.fq -or mason-s3_2.fq -oa mason-s3.sam --illumina-prob-mismatch-scale 2.5 --illumina-read-length 150

tex/minimap2.bib

@@ -61,13 +61,6 @@
 	Volume = {32},
 	Year = {2016}}
 
-@misc{Suzuki:2016,
-	title = {Fast and accurate alignment tool for PacBio and Nanopore long reads},
-	author = {Hajime Suzuki},
-	journal = {Unpublished},
-	howpublished = {\href{https://github.com/ocxtal/minialign}{https://github.com/ocxtal/minialign}},
-	year = {2016}}
-
 @misc{Ruan:2016,
 	title = {Ultra-fast de novo assembler using long noisy reads},
 	author = {Jue Ruan},
@@ -172,14 +165,6 @@
 	Volume = {29},
 	Year = {2011}}
 
-@article {Suzuki130633,
-	author = {Suzuki, Hajime and Kasahara, Masahiro},
-	title = {Acceleration Of Nucleotide Semi-Global Alignment With Adaptive Banded Dynamic Programming},
-	year = {2017},
-	note = {doi:10.1101/130633},
-	publisher = {Cold Spring Harbor Labs Journals},
-	journal = {bioRxiv}}
-
 @article{Gotoh:1982aa,
 	Author = {Gotoh, O},
 	Journal = {J Mol Biol},
@@ -259,3 +244,97 @@
 	Title = {{Striped Smith-Waterman speeds database searches six times over other SIMD implementations}},
 	Volume = {23},
 	Year = {2007}}
+
+@techreport{Holtgrewe:2010aa,
+	Address = {Freie Universit{\"a}t Berlin},
+	Author = {Holtgrewe, M.},
+	Institution = {Institut f{\"u}r Mathematik und Informatik},
+	Number = {TR-B-10-06},
+	Title = {Mason -- a read simulator for second generation sequencing data},
+	Year = {2010}}
+
+@article{Zaharia:2011aa,
+	Author = {Zaharia, Matei and others},
+	Journal = {arXiv:1111:5572},
+	Title = {Faster and More Accurate Sequence Alignment with {SNAP}},
+	Year = {2011}}
+
+@article{Irimia:2008aa,
+	Author = {Irimia, Manuel and Roy, Scott William},
+	Journal = {PLoS Genet},
+	Pages = {e1000148},
+	Title = {Evolutionary convergence on highly-conserved 3' intron structures in intron-poor eukaryotes and insights into the ancestral eukaryotic genome},
+	Volume = {4},
+	Year = {2008}}
+
+@article{Depristo:2011vn,
+	Author = {Depristo, Mark A and others},
+	Journal = {Nat Genet},
+	Pages = {491-8},
+	Title = {A framework for variation discovery and genotyping using next-generation {DNA} sequencing data},
+	Volume = {43},
+	Year = {2011}}
+
+@article{Kurtz:2004zr,
+	Author = {Kurtz, Stefan and others},
+	Journal = {Genome Biol},
+	Pages = {R12},
+	Title = {Versatile and open software for comparing large genomes},
+	Volume = {5},
+	Year = {2004}}
+
+@article {Li223297,
+	author = {Li, Heng and others},
+	title = {New synthetic-diploid benchmark for accurate variant calling evaluation},
+	year = {2017},
+	note = {doi:10.1101/223297},
+	journal = {bioRxiv}
+}
+
+@article{Berlin:2015xy,
+	Author = {Berlin, Konstantin and others},
+	Journal = {Nat Biotechnol},
+	Pages = {623-30},
+	Title = {Assembling large genomes with single-molecule sequencing and locality-sensitive hashing},
+	Volume = {33},
+	Year = {2015}}
+
+@article{Gurevich:2013aa,
+	Author = {Gurevich, Alexey and others},
+	Journal = {Bioinformatics},
+	Pages = {1072-5},
+	Title = {{QUAST}: quality assessment tool for genome assemblies},
+	Volume = {29},
+	Year = {2013}}
+
+@article{Li:2010fk,
+	Author = {Li, Heng and Durbin, Richard},
+	Journal = {Bioinformatics},
+	Pages = {589-95},
+	Title = {Fast and accurate long-read alignment with {Burrows-Wheeler} transform},
+	Volume = {26},
+	Year = {2010}}
+
+@article{Marcais:2018aa,
+	Author = {Mar{\c c}ais, Guillaume and others},
+	Journal = {PLoS Comput Biol},
+	Pages = {e1005944},
+	Title = {{MUMmer4}: A fast and versatile genome alignment system},
+	Volume = {14},
+	Year = {2018}}
+
+@article{Li:2009ys,
+	Author = {Li, Heng and others},
+	Journal = {Bioinformatics},
+	Pages = {2078-9},
+	Title = {The {Sequence Alignment/Map format and SAMtools}},
+	Volume = {25},
+	Year = {2009}}
+
+@article{Suzuki:2018aa,
+	Author = {Suzuki, Hajime and Kasahara, Masahiro},
+	Journal = {BMC Bioinformatics},
+	Pages = {45},
+	Title = {Introducing difference recurrence relations for faster semi-global alignment of long sequences},
+	Volume = {19},
+	Year = {2018}}

tex/minimap2.tex

@@ -1,6 +1,6 @@
 \documentclass{bioinfo}
-\copyrightyear{2017}
-\pubyear{2017}
+\copyrightyear{2018}
+\pubyear{2018}
 
 \usepackage{graphicx}
 \usepackage{hyperref}
@@ -13,29 +13,37 @@
 
 \usepackage{natbib}
 \bibliographystyle{apalike}
-\usepackage{hyperref}
 
 \DeclareMathOperator*{\argmax}{argmax}
 
 \begin{document}
 \firstpage{1}
 
-\title[Aligning long nucleotide sequences with minimap2]{Minimap2: fast pairwise alignment for long nucleotide sequences}
+\title[Aligning nucleotide sequences with minimap2]{Minimap2: pairwise alignment for nucleotide sequences}
 \author[Li]{Heng Li}
 \address{Broad Institute, 415 Main Street, Cambridge, MA 02142, USA}
 
 \maketitle
 
 \begin{abstract}
-\section{Summary:} Minimap2 is a general-purpose mapper to align long noisy DNA
-or mRNA sequences against a large reference database. It targets query
-sequences of 1kb--100Mb in length with per-base divergence typically below
-25\%. For DNA sequence reads, minimap2 is $\sim$30 times faster than many
-mainstream long-read aligners and achieves higher accuracy on simulated data.
-It also employs concave gap cost and rescues inversions for improved alignment
-around potential structural variations. For real long RNA-seq reads, minimap2
-is $\sim$40 times faster than peers and produces alignment more consistent with
-existing gene annotations.
+
+\section{Motivation:} Recent advances in sequencing technologies promise
+ultra-long reads of $\sim$100 kilo bases (kb) in average, full-length mRNA or
+cDNA reads in high throughput and genomic contigs over 100 mega bases (Mb) in
+length. Existing alignment programs are unable or inefficient to process such data
+at scale, which presses for the development of new alignment algorithms.
+
+\section{Results:} Minimap2 is a general-purpose alignment program to map DNA or long
+mRNA sequences against a large reference database. It works with accurate short
+reads of $\ge$100bp in length, $\ge$1kb genomic reads at error rate $\sim$15\%,
+full-length noisy Direct RNA or cDNA reads, and assembly contigs or closely
+related full chromosomes of hundreds of megabases in length. Minimap2 does
+split-read alignment, employs concave gap cost for long insertions and
+deletions (INDELs) and introduces new heuristics to reduce spurious alignments.
+It is 3--4 times as fast as mainstream short-read mappers at comparable
+accuracy, and is $\ge$30 times faster than long-read genomic or cDNA
+mappers at higher accuracy, surpassing most aligners specialized in one type of
+alignment.
 
 \section{Availability and implementation:}
 \href{https://github.com/lh3/minimap2}{https://github.com/lh3/minimap2}
@@ -56,29 +64,38 @@ the thought that 10kb long sequences should be easier to map than 100bp reads
 because we can more effectively skip repetitive regions, which are often the
 bottleneck of short-read alignment. We confirmed our speculation by achieving
 approximate mapping 50 times faster than BWA-MEM~\citep{Li:2016aa}.
-\citet{Suzuki:2016} extended our work with a fast and novel algorithm on
+\citet{Suzuki:2018aa} extended our work with a fast and novel algorithm on
 generating base-level alignment, which in turn inspired us to develop minimap2
-towards higher accuracy and more practical functionality.
+with added functionality.
 
-Both SMRT and ONT have been applied to sequence spliced mRNAs (RNA-seq). While
+Both SMRT and ONT have been applied to the sequencing of spliced mRNAs (RNA-seq). While
 traditional mRNA aligners work~\citep{Wu:2005vn,Iwata:2012aa}, they are not
 optimized for long noisy sequence reads and are tens of times slower than
 dedicated long-read aligners. When developing minimap2 initially for aligning
-genomic DNA only, we realized minor modifications could make it competitive for
-aligning mRNAs as well. Minimap2 is a first RNA-seq aligner specifically
-designed for long noisy reads.
+genomic DNA only, we realized minor modifications could enable the base
+algorithm to map mRNAs as well. Minimap2 becomes a first RNA-seq aligner
+specifically designed for long noisy reads. We have also extended the original
+algorithm to map short reads at a speed faster than several mainstream
+short-read mappers.
+
+In this article, we will describe the minimap2 algorithm and its applications
+to different types of input sequences. We will evaluate the performance and
+accuracy of minimap2 on several simulated and real data sets and demonstrate
+the versatility of minimap2.
 
 \begin{methods}
 \section{Methods}
 
 Minimap2 follows a typical seed-chain-align procedure as is used by most
 full-genome aligners. It collects minimizers~\citep{Roberts:2004fv} of the
-reference sequences and indexes them in a hash table. Then for each query
-sequence, minimap2 takes query minimizers as \emph{seeds}, finds matches to the
-reference, and identifies sets of colinear seeds, which are called
+reference sequences and indexes them in a hash table, with the key being the
+hash of a minimizer and the value being a list of locations of the minimizer
+copies. Then for each query
+sequence, minimap2 takes query minimizers as \emph{seeds}, finds exact matches
+(i.e. \emph{anchors}) to the reference, and identifies sets of colinear anchors as
 \emph{chains}. If base-level alignment is requested, minimap2 applies dynamic
-programming (DP) to extend from the ends of chains and to close unseeded
-regions between adjacent seeds in chains.
+programming (DP) to extend from the ends of chains and to close
+regions between adjacent anchors in chains.
 
 Minimap2 uses indexing and seeding algorithms similar to
 minimap~\citep{Li:2016aa}, and furthers the predecessor with more accurate
@@ -103,9 +120,15 @@ distance between two anchors is too large); otherwise
 \begin{equation}\label{eq:chain-gap}
 \beta(j,i)=\gamma_c\big((y_i-y_j)-(x_i-x_j)\big)
 \end{equation}
-In implementation, a gap of length $l$ costs $\gamma_c(l)=0.01\cdot \bar{w}\cdot
-|l|+0.5\log_2|l|$, where $\bar{w}$ is the average seed length. For $m$ anchors, directly computing all $f(\cdot)$ with
-Eq.~(\ref{eq:chain}) takes $O(m^2)$ time. Although theoretically faster
+In implementation, a gap of length $l$ costs
+\[
+\gamma_c(l)=\left\{\begin{array}{ll}
+0.01\cdot \bar{w}\cdot|l|+0.5\log_2|l| & (l\not=0) \\
+0 & (l=0)
+\end{array}\right.
+\]
+where $\bar{w}$ is the average seed length. For $N$ anchors, directly computing all $f(\cdot)$ with
+Eq.~(\ref{eq:chain}) takes $O(N^2)$ time. Although theoretically faster
 chaining algorithms exist~\citep{Abouelhoda:2005aa}, they
 are inapplicable to generic gap cost, complex to implement and usually
 associated with a large constant. We introduced a simple heuristic to
@@ -115,23 +138,25 @@ We note that if anchor $i$ is chained to $j$, chaining $i$ to a predecessor
 of $j$ is likely to yield a lower score. When evaluating Eq.~(\ref{eq:chain}),
 we start from anchor $i-1$ and stop the process if we cannot find a better
 score after up to $h$ iterations. This approach reduces the average time to
-$O(h\cdot m)$. In practice, we can almost always find the optimal chain with
+$O(hN)$. In practice, we can almost always find the optimal chain with
 $h=50$; even if the heuristic fails, the optimal chain is often close.
 
 \subsubsection{Backtracking}
 Let $P(i)$ be the index of the best predecessor of anchor $i$. It equals 0 if
-$f(i)=w_i$ or $\argmax_j\{f(j)+\eta(j,i)-\gamma(j,i)\}$ otherwise. For each
+$f(i)=w_i$ or $\argmax_j\{f(j)+\alpha(j,i)-\beta(j,i)\}$ otherwise. For each
 anchor $i$ in the descending order of $f(i)$, we apply $P(\cdot)$ repeatedly to
 find its predecessor and mark each visited $i$ as `used', until $P(i)=0$ or we
 reach an already `used' $i$. This way we find all chains with no anchors used
 in more than one chains.
 
-\subsubsection{Identifying primary chains}
+\subsubsection{Identifying primary chains}\label{sec:primary}
 In the absence of copy number changes, each query segment should not be mapped
 to two places in the reference. However, chains found at the previous step may
-have significant or complete overlaps due to repeats in the reference.
+have significant or complete overlaps due to repeats in the reference~\citep{Li:2010fk}.
 Minimap2 used the following procedure to identify \emph{primary chains} that do
-not greatly overlap on the query. Let $Q$ be an empty set initially. For each
+not greatly overlap on the query.
+
+Let $Q$ be an empty set initially. For each
 chain from the best to the worst according to their chaining scores: if on the
 query, the chain overlaps with a chain in $Q$ by 50\% or higher percentage of
 the shorter chain, mark the chain as secondary to the chain in $Q$; otherwise,
@@ -139,7 +164,64 @@ add the chain to $Q$. In the end, $Q$ contains all the primary chains. We did
 not choose a more sophisticated data structure (e.g. range tree or k-d tree)
 because this step is not the performance bottleneck.
 
-\subsection{Aligning genomic DNA}
+For each primary chain, minimap2 estimates its mapping quality with an
+empirical formula:
+\[
+{\rm mapQ}=40\cdot (1-f_2/f_1)\cdot\min\{1,m/10\}\cdot\log f_1
+\]
+where $\log$ denotes natural logarithm, $m$ is the number of anchors on the primary chain, $f_1$ is the chaining
+score, and $f_2\le f_1$ is the score of the best chain that is secondary to the
+primary chain. Intuitively, a chain is assigned to a higher mapping quality if
+it is long and its best secondary chain is weak.
+
+\subsubsection{Estimating per-base sequence divergence}
+Suppose a query sequence harbors $n$ seeds of length $k$, $m$ of which are
+present in a chain. We want to estimate the sequence divergence $\epsilon$
+between the query and the reference sequences in the chain. This is useful
+when base-level alignment is too expensive to perform.
+
+If we model substitutions with a homogeneous Poisson process along the query
+sequence, the probablity of seeing $k$ consecutive bases without substitutions
+is $e^{-k\epsilon}$. On the assumption that all $k$-mers are independent of
+each other, the likelihood function of $\epsilon$ is
+\[
+\mathcal{L}(\epsilon|n,m,k)=e^{-m\cdot k\epsilon}(1-e^{-k\epsilon})^{n-m}
+\]
+The maximum likelihood estimate of $\epsilon$ is
+\[
+\hat{\epsilon}=\frac{1}{k}\log\frac{n}{m}
+\]
+In reality, sequencing errors are sometimes clustered and $k$-mers are not
+independent of each other, especially when we take minimizers as seeds. These
+violate the assumptions in the derivation above. As a result, $\hat{\epsilon}$
+is only approximate and can be biased. It also ignores long deletions from the
+reference sequence. In practice, fortunately, $\hat{\epsilon}$ is often close
+to and strongly correlated with the sequence divergence estimated from
+base-level alignments. On the several datasets used in
+Section~\ref{sec:long-genomic}, the Spearman correlation coefficient is around
+$0.9$.
+
+\subsubsection{Indexing with homopolymer compressed $k$-mers}
+SmartDenovo
+(\href{https://github.com/ruanjue/smartdenovo}{https://github.com/ruanjue/smartdenovo};
+J. Ruan, personal communication) indexes reads with homopolymer-compressed (HPC)
+$k$-mers and finds the strategy improves overlap sensitivity for SMRT reads.
+Minimap2 adopts the same heuristic.
+
+The HPC string of a string $s$, denoted by ${\rm HPC}(s)$, is constructed by
+contracting homopolymers in $s$ to a single base.  An HPC $k$-mer of $s$ is a
+$k$-long substring of ${\rm HPC}(s)$. For example, suppose $s={\tt GGATTTTCCA}$,
+${\rm HPC}(s)={\tt GATCA}$ and the first HPC 4-mer is ${\tt GATC}$.
+
+To demonstrate the effectiveness of HPC $k$-mers, we performed read overlapping
+for the example {\it E. coli} SMRT reads from PBcR~\citep{Berlin:2015xy}, using
+different types of $k$-mers. With normal 15bp minimizers per 5bp window,
+minimap2 finds 90.9\% of $\ge$2kb overlaps inferred from the read-to-reference
+alignment. With HPC 19-mers per 5bp window, minimap2 finds 97.4\% of overlaps. It achieves this
+higher sensitivity by indexing 1/3 fewer minimizers, which further helps
+performance. HPC-based indexing reduces the sensitivity for current ONT reads, though.
+
+\subsection{Aligning genomic DNA}\label{sec:genomic}
 
 \subsubsection{Alignment with 2-piece affine gap cost}
 
@@ -168,7 +250,7 @@ where $s(i,j)$ is the score between the $i$-th reference base and $j$-th query
 base. Eq.~(\ref{eq:ae86}) is a natural extension to the equation under affine
 gap cost~\citep{Gotoh:1982aa,Altschul:1986aa}.
 
-\subsubsection{Suzuki's formulation}
+\subsubsection{The Suzuki-Kasahara formulation}
 
 When we allow gaps longer than several hundred base pairs, nucleotide-level
 alignment is much slower than chaining. SSE acceleration is critical to the
@@ -176,14 +258,14 @@ performance of minimap2. Traditional SSE implementations~\citep{Farrar:2007hs}
 based on Eq.~(\ref{eq:ae86}) can achieve 16-way parallelization for short
 sequences, but only 4-way parallelization when the peak alignment score reaches
 32767. Long sequence alignment may exceed this threshold. Inspired by
-\citet{Wu:1996aa} and the following work, \citet{Suzuki:2016} proposed a
-difference-based formulation that lifted this limitation. In case of 2-piece
-gap cost, define
+\citet{Wu:1996aa} and the following work, \citet{Suzuki:2018aa} proposed a
+difference-based formulation that lifted this limitation.
+In case of 2-piece gap cost, define
 \[
 \left\{\begin{array}{ll}
 u_{ij}\triangleq H_{ij}-H_{i-1,j} & v_{ij}\triangleq H_{ij}-H_{i,j-1} \\
-x_{ij}\triangleq E_{i+1,j}-H_{ij} & \tilde{x}_{ij}\triangleq \tilde{E}_{i+1,j}-\tilde{H}_{ij} \\
-y_{ij}\triangleq F_{i,j+1}-H_{ij} & \tilde{y}_{ij}\triangleq \tilde{F}_{i,j+1}-\tilde{H}_{ij}
+x_{ij}\triangleq E_{i+1,j}-H_{ij} & \tilde{x}_{ij}\triangleq \tilde{E}_{i+1,j}-H_{ij} \\
+y_{ij}\triangleq F_{i,j+1}-H_{ij} & \tilde{y}_{ij}\triangleq \tilde{F}_{i,j+1}-H_{ij}
 \end{array}\right.
 \]
 We can transform Eq.~(\ref{eq:ae86}) to
@@ -199,9 +281,10 @@ y_{ij}&=&\max\{0,y_{i,j-1}+u_{i,j-1}-z_{ij}+q\}-q-e\\
 \tilde{y}_{ij}&=&\max\{0,\tilde{y}_{i,j-1}+u_{i,j-1}-z_{ij}+\tilde{q}\}-\tilde{q}-\tilde{e}
 \end{array}\right.
 \end{equation}
-where $z_{ij}$ is a temporary variable that does not need to be stored.  An
-important property of Eq.~(\ref{eq:suzuki}) is that all values are bounded. To
-see that,
+where $z_{ij}$ is a temporary variable that does not need to be stored.
+
+An important property of Eq.~(\ref{eq:suzuki}) is that all values are bounded
+by scoring parameters. To see that,
 \[
 x_{ij}=E_{i+1,j}-H_{ij}=\max\{-q,E_{ij}-H_{ij}\}-e
 \]
@@ -242,11 +325,11 @@ y_{rt}&=&\max\{0,y_{r-1,t}+u_{r-1,t}-z_{rt}+q\}-q-e\\
 \end{equation*}
 In this formulation, cells with the same diagonal index $r$ are independent of
 each other. This allows us to fully vectorize the computation of all cells on
-the same anti-diagonal in one inner loop. It also simplifies banded alignment,
+the same anti-diagonal in one inner loop. It also simplifies banded alignment (500bp band width by default),
 which would be difficult with striped vectorization~\citep{Farrar:2007hs}.
 
-On the condition that $q+e<\tilde{q}+\tilde{e}$ and $e>\tilde{e}$, the boundary
-condition of the equation above is
+On the condition that $q+e<\tilde{q}+\tilde{e}$ and $e>\tilde{e}$, the initial
+values in the diagonal-antidiagonal formuation are
 \[
 \left\{\begin{array}{l}
 x_{r-1,-1}=y_{r-1,r}=-q-e\\
@@ -263,14 +346,21 @@ r\cdot(e-\tilde{e})-(\tilde{q}-q)-\tilde{e} & (r=\lceil\frac{\tilde{q}-q}{e-\til
 -\tilde{e} & (r>\lceil\frac{\tilde{q}-q}{e-\tilde{e}}-1\rceil)
 \end{array}\right.
 \]
-These can be derived from the initial conditions of Eq.~(\ref{eq:ae86}).
+These can be derived from the initial values for Eq.~(\ref{eq:ae86}).
+
+When performing global alignment, we do not need to compute $H_{rt}$ in each cell.
+We use 16-way vectorization throughout the alignment process. When extending
+alignments from ends of chains, we need to find the cell $(r,t)$ where $H_{rt}$
+reaches the maximum. We resort to 4-way vectorization to compute
+$H_{rt}=H_{r-1,t}+u_{rt}$. Because this computation is simple,
+Eq.~(\ref{eq:suzuki}) is still the dominant performance bottleneck.
 
 In practice, our 16-way vectorized implementation of global alignment is three
 times as fast as Parasail's 4-way vectorization~\citep{Daily:2016aa}.  Without
 banding, our implementation is slower than Edlib~\citep{Sosic:2017aa}, but with
 a 1000bp band, it is considerably faster. When performing global alignment
 between anchors, we expect the alignment to stay close to the diagonal of the
-DP matrix. Banding is applicable most of time.
+DP matrix. Banding is applicable most of the time.
 
 \subsubsection{The Z-drop heuristic}
 
@@ -285,15 +375,25 @@ $j'<j$, such that
 S(i',j')-S(i,j)>Z+e\cdot|(i-i')-(j-j')|
 \]
 where $e$ is the gap extension cost and $Z$ is an arbitrary threshold.
-This strategy is similar to X-drop employed in BLAST~\citep{Altschul:1997vn}.
-However, unlike X-drop, it would not break the alignment in the presence of a
-single long gap.
+This strategy is first used in BWA-MEM. It is similar to X-drop employed in
+BLAST~\citep{Altschul:1997vn}, but unlike X-drop, it would not break the
+alignment in the presence of a single long gap.
 
 When minimap2 breaks a global alignment between two anchors, it performs local
 alignment between the two subsequences involved in the global alignment, but
 this time with the one subsequence reverse complemented. This additional
 alignment step may identify short inversions that are missed during chaining.
 
+\subsubsection{Filtering out misplaced anchors}
+Due to sequencing errors and local homology, some anchors in a chain may be
+wrong. If we blindly align regions between two misplaced anchors, we will
+produce a suboptimal alignment. To reduce this artifact, we filter out
+anchors that lead to a $>$10bp insertion and a $>$10bp deletion at the same
+time, and filter out terminal anchors that lead to a long gap towards the ends
+of a chain. These heuristics greatly alleviate the issues with misplaced
+anchors, but they are unable to fix all such errors. Local misalignment is a
+limitation of minimap2 which we hope to address in future.
+
 \subsection{Aligning spliced sequences}
 
 The algorithm described above can be adapted to spliced alignment. In this
@@ -325,18 +425,24 @@ F_{i,j+1}= \max\{H_{ij}-q,F_{ij}\}-e\\
 \tilde{E}_{i+1,j}= \max\{H_{ij}-d(i)-\tilde{q},\tilde{E}_{ij}\}\\
 \end{array}\right.
 \end{equation}
-Let $T$ be the reference sequence. $d(i)$ is the cost of a non-canonical donor
-site, which takes 0 if $T[i+1,i+2]={\tt GT}$, or a postive number $p$
-otherwise. Similarly, $a(i)$ is the cost of a non-canonical acceptor site, which
-takes 0 if $T[i-1,i]={\tt AG}$, or $p$ otherwise. Eq.~(\ref{eq:splice}) is
-almost equivalent to the equation used by EXALIN~\citep{Zhang:2006aa} except
-that we allow insertions immediately followed by deletions and vice versa; in
-addition, we use Suzuki's diagonal formulation in actual implementation.
-
-%Given that $d_i$ and $a_i$
-%are a function of the reference sequence, it is possible to incorporate
-%splicing signals with more sophisticated models, such as positional weight
-%matrices. We have not tried this approach.
+Let $T$ be the reference sequence. $d(i)$ is computed as
+\[d(i)=\left\{\begin{array}{ll}
+0 & \mbox{if $T[i+1,i+3]$ is ${\tt GTA}$ or ${\tt GTG}$} \\
+p/2 & \mbox{if $T[i+1,i+3]$ is ${\tt GTC}$ or ${\tt GTT}$} \\
+p & \mbox{otherwise}
+\end{array}\right.\]
+where $T[i,j]$ extracts a substring of $T$ between $i$ and $j$ inclusively.
+$d(i)$ penalizes non-canonical donor sites with $p$ and less frequent Eukaryotic
+splicing signal ${\tt GT[C/T]}$ with $p/2$~\citep{Irimia:2008aa}. Similarly,
+\[a(i)=\left\{\begin{array}{ll}
+0 & \mbox{if $T[i-2,i]$ is ${\tt CAG}$ or ${\tt TAG}$} \\
+p/2 & \mbox{if $T[i-2,i]$ is ${\tt AAG}$ or ${\tt GAG}$} \\
+p & \mbox{otherwise}
+\end{array}\right.\]
+models the acceptor signal. Eq.~(\ref{eq:splice}) is close to an equation in
+\citet{Zhang:2006aa} except that we allow insertions immediately followed by
+deletions and vice versa; in addition, we use the Suzuki-Kasahara diagonal
+formulation in actual implementation.
 
 If RNA-seq reads are not sequenced from stranded libraries, the read strand
 relative to the underlying transcript is unknown. By default, minimap2 aligns
@@ -348,31 +454,65 @@ reads that span canonical splicing sites.
 
 In the spliced alignment mode, minimap2 further increases the density of
 minimizers and disables banded alignment. Together with the two-round DP-based
-alignment, spliced alignment is several times slower than DNA sequence
+alignment, spliced alignment is several times slower than genomic DNA
 alignment.
 
+\subsection{Aligning short paired-end reads}
+
+During chaining, minimap2 takes a pair of reads as one fragment with a gap of
+unknown length in the middle. It applies a normal gap cost between seeds on the
+same read but is a more permissive gap cost between seeds on different reads.
+More precisely, the gap cost during chaining is ($l\not=0$):
+\[
+\gamma_c(l)=\left\{\begin{array}{ll}
+0.01\cdot\bar{w}\cdot |l|+0.5\log_2 |l| & \mbox{if two seeds on the same read} \\
+\min\{0.01\cdot\bar{w}\cdot|l|,\log_2|l|\} & \mbox{otherwise}
+\end{array}\right.
+\]
+After identifying primary chains (Section~\ref{sec:primary}), we split each
+fragment chain into two read chains and perform alignment for each read as in
+Section~\ref{sec:genomic}.  Finally, we pair hits of each read end to find
+consistent paired-end alignments.
+
 \end{methods}
 
 \section{Results}
 
-\subsection{Aligning genomic reads}
+Minimap2 is implemented in the C programming language and comes with APIs in
+both C and Python. It is distributed under the MIT license, free to both
+commercial and academic uses. Minimap2 uses the same base algorithm for all
+applications, but it has to apply different sets of parameters depending on
+input data types. Similar to BWA-MEM, minimap2 introduces `presets' that
+modify multiple parameters with a simple invocation. Detailed settings
+and command-line options can be found in the minimap2 manpage. In addition to
+the applications evaluated in the following sections, minimap2 also retains
+minimap's functionality to find overlaps between long reads and to search
+against large multi-species databases such as \emph{nt} from NCBI.
+
+\subsection{Aligning long genomic reads}\label{sec:long-genomic}
 
 \begin{figure}[!tb]
 \centering
 \includegraphics[width=.5\textwidth]{roc-color.pdf}
-\caption{Evaluation on simulated SMRT reads aligned against human genome
-GRCh38. (a) ROC-like curve. Alignments are sorted by mapping quality in the
-descending order. For each mapping quality threshold, the fraction of
-alignments with mapping quality above the threshold and their error rate
-are plotted. (b) Accumulative mapping error rate as a function of mapping
-quality. 33,088 $\ge$1000bp reads were simulated using pbsim~\citep{Ono:2013aa}
-with error profile sampled from file `m131017\_060208\_42213\_*.1.*' downloaded
-at \href{http://bit.ly/chm1p5c3}{http://bit.ly/chm1p5c3}. The N50 read length
-is 11,628. A read is considered correctly mapped if the true position overlaps
-with the best mapping position by 10\% of the read length. All aligners were
-run under the default setting for SMRT reads. Kart outputted all alignments at
-mapping quality 60, so is not shown in the figure. It mapped nearly all reads
-with 4.1\% of alignments being wrong, less accurate than others.}\label{fig:eval}
+\caption{Evaluation on aligning simulated reads. Simulated reads were mapped 
+to the primary assembly of human genome GRCh38. A read is considered correctly
+mapped if its longest alignment overlaps with the true interval, and the
+overlap length is $\ge$10\% of the true interval length.  Read alignments are
+sorted by mapping quality in the descending order. For each mapping quality
+threshold, the fraction of alignments (out of the number of input reads) with
+mapping quality above the threshold and their error rate are
+plotted along the curve. (a) long-read alignment evaluation. 33,088 $\ge$1000bp
+reads were simulated using pbsim~\citep{Ono:2013aa} with error profile sampled
+from file `m131017\_060208\_42213\_*.1.*' downloaded at
+\href{http://bit.ly/chm1p5c3}{http://bit.ly/chm1p5c3}. The N50 read length is
+11,628. Aligners were run under the default setting for SMRT reads.
+Kart outputted all alignments at mapping quality 60, so is not shown in the
+figure. It mapped nearly all reads with 4.1\% of alignments being wrong, less
+accurate than others.  (b) short-read alignment evaluation. 10 million pairs of
+150bp reads were simulated using mason2~\citep{Holtgrewe:2010aa} with option
+`\mbox{--illumina-prob-mismatch-scale 2.5}'. Short-read aligners were run under
+the default setting except for changing the maximum fragment length to
+800bp.}\label{fig:eval}
 \end{figure}
 
 As a sanity check, we evaluated minimap2 on simulated human reads along with
@@ -380,22 +520,20 @@ BLASR~(v1.MC.rc64; \citealp{Chaisson:2012aa}),
 BWA-MEM~(v0.7.15; \citealp{Li:2013aa}),
 GraphMap~(v0.5.2; \citealp{Sovic:2016aa}),
 Kart~(v2.2.5; \citealp{Lin:2017aa}),
-minialign~(v0.5.3; \citealp{Suzuki:2016}) and
+minialign~(v0.5.3; \href{https://github.com/ocxtal/minialign}{https://github.com/ocxtal/minialign}) and
 NGMLR~(v0.2.5; \citealp{Sedlazeck169557}). We excluded rHAT~\citep{Liu:2016ab}
 and LAMSA~\citep{Liu:2017aa} because they either
-crashed or produced malformatted output. In this evaluation, Minimap2 has
+crashed or produced malformatted output. In this evaluation, minimap2 has
 higher power to distinguish unique and repetitive hits, and achieves overall
-higher mapping accuracy (Fig.~\ref{fig:eval}a). It is still the most accurate
-even if we skip DP-based alignment (data not shown), confirming chaining alone
-is sufficient to achieve high accuracy for approximate mapping. Minimap2 and
+higher mapping accuracy (Fig.~\ref{fig:eval}a). Minimap2 and
 NGMLR provide better mapping quality estimate: they rarely give repetitive hits
-high mapping quality (Fig.~\ref{fig:eval}b).  Apparently, other aligners may
+high mapping quality.  Apparently, other aligners may
 occasionally miss close suboptimal hits and be overconfident in wrong mappings.
-On run time, minialign is slightly faster than minimap2 and Kart. They are over
-30 times faster than the rest.  Minimap2 consumed 6.1GB memory at the peak,
-more than BWA-MEM but less than others.
+On run time, minimap2 took 200 CPU seconds, comparable to minialign and Kart, and is over
+30 times faster than the rest.  Minimap2 consumed 6.8GB memory at the peak,
+more than BWA-MEM (5.4GB), similar to NGMLR and less than others.
 
-On real human SMRT reads, the relative performance and sensitivity of
+On real human SMRT reads, the relative performance and fraction of mapped reads reported by
 these aligners are broadly similar to the metrics on simulated data. We are
 unable to provide a good estimate of mapping error rate due to the lack of the
 truth.  On ONT $\sim$100kb human reads~\citep{Jain128835}, BWA-MEM failed.
@@ -405,19 +543,19 @@ confirm the observation by~\citet{Sedlazeck169557} that BWA-MEM often breaks
 them into shorter gaps. The issue is much alleviated with minimap2, thanks
 to the 2-piece affine gap cost.
 
-\subsection{Aligning spliced reads}
+\subsection{Aligning long spliced reads}
 
 We evaluated minimap2 on SIRV control data~(AC:SRR5286959;
-\citealp{Byrne:2017aa}) where the truth is known. Minimap2 predicted 59\,916
-introns from 11\,017 reads. 93.0\% of splice juctions are precise. We examined
+\citealp{Byrne:2017aa}) where the truth is known. Minimap2 predicted 59\,918
+introns from 11\,018 reads. 93.8\% of splice juctions are precise. We examined
 wrongly predicted junctions and found the majority were caused by clustered
 splicing signals (e.g. two adjacent ${\tt GT}$ sites). When INDEL sequencing
 errors are frequent, it is difficult to find precise splicing sites in this
 case. If we allow up to 10bp distance from true splicing sites, 98.4\% of
-aligned introns are approximately correct.  Given this observation, we might be
-able to improve boundary detection by initializing $d(\cdot)$ and $a(\cdot)$ in
-Eq.~(\ref{eq:splice}) with position-specific scoring matrices or more
-sophisticated models. We have not tried this approach.
+aligned introns are approximately correct. It is worth noting that for SIRV, we
+asked minimap2 to model the ${\tt GT..AG}$ splicing signal only without extra
+bases. This is because SIRV does not honor the evolutionarily prevalent signal
+${\tt GT[A/G]..[C/T]AG}$~\citep{Irimia:2008aa}.
 
 \begin{table}[!tb]
 \processtable{Evaluation of junction accuracy on 2D ONT reads}
@@ -426,18 +564,18 @@ sophisticated models. We have not tried this approach.
 \toprule
 & GMAP & minimap2 & SpAln & STAR\\
 \midrule
-Run time (CPU min) & 631 & 15.5 & 2\,076 & 33.9 \\
-Peak RAM (GByte) & 8.9 & 14.5 & 3.2 & 29.2\vspace{1em}\\
-\# aligned reads & 103\,669 & 103\,917 & 103\,711 & 26\,479\\
-\# chimeric alignments & 1\,904 & 1\,671 & 0 & 0\\
-\# non-spliced alignments & 15\,854 & 14\,483 & 17\,033 & 10\,545\vspace{1em}\\
-\# aligned introns & 692\,275 & 694\,237 & 692\,945 & 78\,603 \\
-\# novel introns & 11\,239 & 3\,217 & 8\,550 & 1\,214 \\
-\% exact introns & 83.8\% & 91.8\% & 87.9\% & 55.2\% \\
-\% approx. introns & 91.8\% & 96.5\% & 92.5\% & 82.4\% \\
+Run time (CPU min)        & 631      & 15.9     & 2\,076   & 33.9 \\
+Peak RAM (GByte)          & 8.9      & 14.5     & 3.2      & 29.2\vspace{1em}\\
+\# aligned reads          & 103\,669 & 104\,199 & 103\,711 & 26\,479 \\
+\# chimeric alignments    & 1\,904   & 1\,488   & 0        & 0 \\
+\# non-spliced alignments & 15\,854  & 14\,798  & 17\,033  & 10\,545\vspace{1em}\\
+\# aligned introns        & 692\,275 & 693\,553 & 692\,945 & 78\,603 \\
+\# novel introns          & 11\,239  & 3\,113   & 8\,550   & 1\,214 \\
+\% exact introns          & 83.8\%   & 94.0\%   & 87.9\%   & 55.2\% \\
+\% approx. introns        & 91.8\%   & 96.9\%   & 92.5\%   & 82.4\% \\
 \botrule
 \end{tabular}
-}{Mouse reads (AC:SRR5286960) were mapped to the primary assembly of mouse
+}{Mouse cDNA reads (AC:SRR5286960; R9.4 chemistry) were mapped to the primary assembly of mouse
 genome GRCm38 with the following tools and command options: minimap2 (`-ax
 splice'); GMAP (`-n 0 --min-intronlength 30 --cross-species'); SpAln (`-Q7 -LS
 -S3'); STARlong (according to
@@ -446,7 +584,7 @@ compared to the EnsEMBL gene annotation, release 89. A predicted intron
 is \emph{novel} if it has no overlaps with any annotated introns. An intron
 is \emph{exact} if it is identical to an annotated intron. An intron is
 \emph{approximate} if both its 5'- and 3'-end are within 10bp around the ends
-of an annotated intron.}
+of an annotated intron. Chimeric alignments are defined in the SAM spec~\citep{Li:2009ys}.}
 \end{table}
 
 We next aligned real mouse reads~\citep{Byrne:2017aa} with GMAP~(v2017-06-20;
@@ -455,10 +593,20 @@ STAR~(v2.5.3a; \citealp{Dobin:2013kx}). In general, minimap2 is more
 consistent with existing annotations (Table~\ref{tab:intron}): it finds
 more junctions with a higher percentage being exactly or approximately correct.
 Minimap2 is over 40 times faster than GMAP and SpAln. While STAR is close to
-minimap2 in speed, it does not work well with noisy reads.  We have also
-evaluated spliced aligners on public Iso-Seq data (human Alzheimer brain
-from \href{http://bit.ly/isoseqpub}{http://bit.ly/isoseqpub}). The observation
-is similar: minimap2 is faster at higher junction accuracy.
+minimap2 in speed, it does not work well with noisy reads.
+
+We have also evaluated spliced aligners on a human Nanopore Direct RNA-seq
+dataset (\href{http://bit.ly/na12878ont}{http://bit.ly/na12878ont}). Minimap2
+aligned 10 million reads in $<$1 wall-clock hour using 16 CPU cores. 94.2\% of
+aligned splice junctions consistent with gene annotations. In comparison,
+GMAP under option `-k 14 -n 0 --min-intronlength 30 --cross-species' is 160
+times slower; 68.7\% of GMAP junctions are found in known gene annotations. The
+percentage increases to 84.1\% if an aligned junction within 10bp from an
+annotated junction is considered to be correct. On a public Iso-Seq dataset
+(human Alzheimer brain from
+\href{http://bit.ly/isoseqpub}{http://bit.ly/isoseqpub}), minimap2 is also
+faster at higher junction accuracy in comparison to other aligners in
+Table~\ref{tab:intron}.
 
 We noted that GMAP and SpAln have not been optimized for noisy reads. We are
 showing the best setting we have experimented, but their developers should be
@@ -469,39 +617,107 @@ able to improve their accuracy further.
 %{\footnotesize
 %\begin{tabular}{lrrrr}
 %\toprule
-%& GMAP & minimap2 & SpAln & STAR\\
+%                          &      GMAP &  minimap2 &     SpAln &      STAR \\ % one GMAP thread took 14 days to align a tiny fraction of reads
 %\midrule
-%Run time (CPU min) & & 243 & 2\,352 & 1\,647 \\
-%\# aligned reads & & 1\,123\,025 & 1\,094\,092 & 682\,452\\
-%\# chimeric alignments & & 33\,091 & 0 & 0\\
-%\# non-spliced alignments & & 339\,081 & 291\,447 & 272\,536\vspace{1em}\\
-%\# aligned introns & & 9\,071\,755 & 9\,208\,564 & 3\,029\,121 \\
-%\# novel introns & & 42\,773 & 82\,230 & 17\,791 \\
-%\% exact introns & & 94.9\% & 91.7\% & 84.7\% \\
-%\% approx. introns&& 96.9\% & 93.4\% & 93.8\% \\
+%Run time (CPU min)        &         - &       243 &     2,352 &     1,647 \\
+%\# aligned reads          & 1,113,502 & 1,123,025 & 1,094,092 &   682,452 \\
+%\# chimeric alignments    &    48,927 &    33,091 &         0 &         0 \\
+%\# non-spliced alignments &   334,097 &   339,081 &   291,447 &   272,536 \vspace{1em}\\
+%\# aligned introns        & 8,922,221 & 9,071,755 & 9,208,564 & 3,029,121 \\
+%\# novel introns          &    48,927 &    42,773 &    82,230 &    17,791 \\
+%\% exact introns          &    90.6\% &    94.9\% &    91.7\% &    84.7\% \\
+%\% approx. introns        &    94.0\% &    96.9\% &    93.4\% &    93.8\% \\
 %\botrule
 %\end{tabular}
 %}{}
 %\end{table}
 
+\subsection{Aligning short genomic reads}
 
-\section{Conclusion}
+We evaluated minimap2 along with Bowtie2~(v2.3.3; \citealt{Langmead:2012fk}), BWA-MEM and
+SNAP (v1.0beta23; \citealt{Zaharia:2011aa}). Minimap2 is 3--4 times as fast as Bowtie2 and
+BWA-MEM, but is 1.3 times slower than SNAP. Minimap2 is more accurate on this
+simulated data set than Bowtie2 and SNAP but less accurate than BWA-MEM
+(Fig.~\ref{fig:eval}b). Closer investigation reveals that BWA-MEM achieves
+a higher accuracy partly because it tries to locally align a read in a small
+region close to its mate. If we disable this feature, BWA-MEM becomes slightly
+less accurate than minimap2. We might implement a similar heuristic
+in minimap2 in future.
 
-Minimap2 is a fast, accurate and versatile aligner for long nucleotide
-sequences.  In addition to reference-based read mapping, minimap2 inherits
-minimap's functionality to search against huge multi-species databases and to
-find read overlaps. On a few test data sets, minimap2 appears to yield slightly
-better miniasm assembly~\citep{Li:2016aa}. Minimap2 can also align similar
-genomes or different assemblies of the same species. However, full-genome
-alignment is an intricate research topic. More thorough evaluations would be
-necessary to justify the use of minimap2 for such applications.
+To evaluate the accuracy of minimap2 on real data, we aligned human reads
+(AC:ERR1341796) with BWA-MEM and minimap2, and called SNPs and small INDELs
+with GATK HaplotypeCaller v3.5~\citep{Depristo:2011vn}. This run was sequenced
+from experimentally mixed CHM1 and CHM13 cell lines. Both of them are homozygous
+across the whole genome and have been \emph{de novo} assembled with SMRT reads
+to high quality. This allowed us to construct an independent truth variant
+dataset~\citep{Li223297} for
+ERR1341796. In this evaluation, minimap2 has higher SNP false negative rate
+(FNR; 2.6\% of minimap2 vs 2.3\% of BWA-MEM), but fewer false positive SNPs per
+million bases (FPPM; 7.0 vs 8.8), similar INDEL FNR (11.2\% vs 11.3\%) and
+similar INDEL FPPM (6.4 vs 6.5). Minimap2 is broadly comparable to BWA-MEM in the
+context of small variant calling.
+
+\subsection{Aligning long-read assemblies}
+
+Minimap2 can align a SMRT assembly (AC:GCA\_001297185.1) against GRCh38 in 7
+minutes using 8 CPU cores, over 20 times faster than nucmer from
+MUMmer4~\citep{Marcais:2018aa}. With the paftools.js script from the minimap2
+package, we called 2.67 million single-base substitutions out of 2.78Gbp
+genomic regions. The transition-to-transversion ratio (ts/tv) is 2.01. In
+comparison, using MUMmer4's dnadiff pipeline, we called 2.86 million
+substitutions in 2.83Gbp at ts/tv=1.87. Given that ts/tv averaged across the
+human genome is about 2 but ts/tv averaged over random errors is 0.5, the
+minimap2 callset arguably has higher precision at lower sensitivity.
+
+The sample being assembled is a female. Minimap2 still called 201 substitutions
+on the Y chromosome. These substitutions all come from one contig aligned at
+96.8\% sequence identity. The contig could be a segmental duplication
+absent from GRCh38. In constrast, dnadiff called 9070 substitutions on the Y
+chromosome across 73 SMRT contigs. This again implies our minimap2-based
+pipeline has higher precision.
+
+\section{Discussions}
+
+Minimap2 is a versatile mapper and pairwise aligner for nucleotide sequences.
+It works with short reads, assembly contigs and long noisy genomic and RNA-seq
+reads, and can be used as a read mapper, long-read overlapper or a full-genome
+aligner. Minimap2 is also accurate and efficient, often outperforming other
+domain-specific alignment tools in terms of both speed and accuracy.
+
+The capability of minimap2 comes from a fast base-level alignment algorithm and
+an accurate chaining algorithm. When aligning long query sequences, base-level
+alignment is often the performance bottleneck. The Suzuki-Kasahara algorithm
+greatly alleviates the bottleneck and enables DP-based splice alignment
+involving $>$100kb introns, which was impractically slow ten years ago.  The
+minimap2 chaining algorithm is fast and highly accurate by itself.  In fact,
+chaining alone is more accurate than all the other long-read mappers in
+Fig.~\ref{fig:eval}a (data not shown). This accuracy helps to reduce downstream
+base-level alignment of candidate chains, which is still several times slower than
+chaining even with the Suzuki-Kasahara improvement. In addition, taking a
+general form, minimap2 chaining can be adapted to non-typical data types such as
+spliced reads and multiple reads per fragment. This gives us the opportunity to
+extend the same base algorithm to a variety of use cases.
+
+Modern mainstream aligners often use a full-text index, such as suffix array or
+FM-index, to index reference sequences. An advantage of this approach is that
+we can use exact seeds of arbitrary lengths, which helps to increase seed
+uniqueness and reduce unsuccessful extensions. Minimap2 indexes reference
+k-mers with a hash table instead. Such fixed-length seeds are inferior to
+variable-length seeds in theory, but can be computed much more efficiently in
+practice. When a query sequence has multiple seed hits, we can afford to skip
+highly repetitive seeds without affecting the final accuracy. This further
+alleviates the concern with the seeding uniqueness. At the same time, at low
+sequence identity, it is rare to see long seeds anyway. Hash table is the ideal
+data structure for mapping long noisy sequences.
 
 \section*{Acknowledgements}
-We owe a debt of gratitude to Hajime Suzuki for releasing his masterpiece and
-insightful notes before formal publication. We thank M. Schatz, P. Rescheneder
-and F.  Sedlazeck for pointing out the limitation of BWA-MEM. We are also
-grateful to early minimap2 testers who have greatly helped to fix various
-issues.
+We owe a debt of gratitude to H. Suzuki and M. Kasahara for releasing their
+masterpiece and insightful notes before formal publication. We thank M.
+Schatz, P. Rescheneder and F. Sedlazeck for pointing out the limitation of
+BWA-MEM. We are also grateful to minimap2 users who have greatly helped to
+suggest features and to fix various issues.
+
+\paragraph{Funding\textcolon} NHGRI 1R01HG010040-01
 
 \bibliography{minimap2}
 

tex/mm2-s3.sam.eval

@@ -0,0 +1,62 @@
+Q	60	18579866	27	0.000001453	18579866
+Q	59	27087	4	0.000001666	18606953
+Q	58	21435	1	0.000001718	18628388
+Q	57	45663	3	0.000001874	18674051
+Q	56	36031	2	0.000001978	18710082
+Q	55	18499	2	0.000002082	18728581
+Q	54	14754	2	0.000002187	18743335
+Q	53	25541	2	0.000002291	18768876
+Q	52	26397	5	0.000002554	18795273
+Q	51	15090	3	0.000002711	18810363
+Q	50	13425	11	0.000003294	18823788
+Q	49	15175	2	0.000003397	18838963
+Q	48	19407	4	0.000003606	18858370
+Q	47	11538	16	0.000004452	18869908
+Q	46	12558	17	0.000005349	18882466
+Q	45	40362	28	0.000006817	18922828
+Q	44	10465	13	0.000007500	18933293
+Q	43	10098	20	0.000008552	18943391
+Q	42	10682	19	0.000009549	18954073
+Q	41	9823	11	0.000010125	18963896
+Q	40	9685	16	0.000010963	18973581
+Q	39	10273	18	0.000011905	18983854
+Q	38	9515	18	0.000012847	18993369
+Q	37	9474	27	0.000014261	19002843
+Q	36	10430	25	0.000015568	19013273
+Q	35	9241	34	0.000017348	19022514
+Q	34	9162	31	0.000018968	19031676
+Q	33	10164	49	0.000021532	19041840
+Q	32	9152	55	0.000024408	19050992
+Q	31	9252	35	0.000026233	19060244
+Q	30	9872	55	0.000029103	19070116
+Q	29	8938	65	0.000032496	19079054
+Q	28	8951	73	0.000036306	19088005
+Q	27	9949	95	0.000041261	19097954
+Q	26	9784	97	0.000046316	19107738
+Q	25	10126	97	0.000051366	19117864
+Q	24	11260	123	0.000057765	19129124
+Q	23	10047	114	0.000063691	19139171
+Q	22	9661	123	0.000070083	19148832
+Q	21	10339	168	0.000078813	19159171
+Q	20	17928	193	0.000088804	19177099
+Q	19	9842	193	0.000098817	19186941
+Q	18	14737	247	0.000111605	19201678
+Q	17	10218	238	0.000123934	19211896
+Q	16	10271	242	0.000136457	19222167
+Q	15	12241	333	0.000153683	19234408
+Q	14	9189	336	0.000171070	19243597
+Q	13	9493	515	0.000197734	19253090
+Q	12	11502	743	0.000236185	19264592
+Q	11	8211	507	0.000262390	19272803
+Q	10	9133	606	0.000293695	19281936
+Q	9	10014	931	0.000341801	19291950
+Q	8	8436	698	0.000377816	19300386
+Q	7	8443	705	0.000414163	19308829
+Q	6	10203	944	0.000462808	19319032
+Q	5	6936	756	0.000501760	19325968
+Q	4	6732	843	0.000545190	19332700
+Q	3	8215	1104	0.000602040	19340915
+Q	2	21201	5440	0.000882342	19362116
+Q	1	82328	22186	0.002019600	19444444
+Q	0	553853	371953	0.020562901	19998297
+U	1703

tex/mm2.approx.eval

@@ -1,30 +1,12 @@
-Q	60	32066	0	0.000000000
-Q	40	32	1	0.000031155
-Q	38	19	1	0.000062272
-Q	36	11	1	0.000093376
-Q	35	32	1	0.000124378
-Q	33	15	1	0.000155400
-Q	32	58	1	0.000186145
-Q	27	11	1	0.000217095
-Q	26	80	1	0.000247494
-Q	21	19	2	0.000309186
-Q	20	16	1	0.000339936
-Q	19	19	1	0.000370622
-Q	18	22	2	0.000432099
-Q	17	37	5	0.000585751
-Q	15	24	2	0.000646930
-Q	14	18	3	0.000738939
-Q	13	30	6	0.000922821
-Q	12	18	1	0.000953054
-Q	11	29	2	0.001013638
-Q	10	30	1	0.001043393
-Q	9	20	5	0.001196099
-Q	8	25	8	0.001440348
-Q	7	28	6	0.001622830
-Q	6	35	12	0.001988132
-Q	5	34	12	0.002352725
-Q	4	29	8	0.002594865
-Q	3	36	14	0.003018937
-Q	2	46	15	0.003471482
-Q	1	69	36	0.004558162
-Q	0	167	94	0.007377173
+Q	60	32084	0	0.000000000	32084
+Q	24	318	2	0.000061725	32402
+Q	11	98	2	0.000123077	32500
+Q	8	37	2	0.000184405	32537
+Q	7	37	3	0.000276294	32574
+Q	6	40	3	0.000367940	32614
+Q	5	34	2	0.000428816	32648
+Q	4	37	5	0.000581306	32685
+Q	3	28	6	0.000764222	32713
+Q	2	38	6	0.000946536	32751
+Q	1	50	21	0.001585318	32801
+Q	0	286	150	0.006105117	33087

tex/mm2.eval

@@ -1,17 +1,13 @@
-Q	60	32072	0	0.000000000
-Q	43	206	1	0.000030981
-Q	27	201	1	0.000061578
-Q	15	59	1	0.000092200
-Q	12	25	1	0.000122839
-Q	11	16	1	0.000153473
-Q	10	24	1	0.000184032
-Q	9	17	2	0.000245248
-Q	8	27	3	0.000336938
-Q	7	23	1	0.000367309
-Q	6	20	1	0.000397675
-Q	5	18	4	0.000519751
-Q	4	17	1	0.000550038
-Q	3	29	5	0.000702204
-Q	2	32	4	0.000823522
-Q	1	54	6	0.001004872
-Q	0	234	106	0.004202697
+Q	60	32477	0	0.000000000	32477
+Q	22	16	1	0.000030776	32493
+Q	21	44	1	0.000061468	32537
+Q	19	73	1	0.000091996	32610
+Q	14	66	1	0.000122414	32676
+Q	10	26	3	0.000214054	32702
+Q	8	14	1	0.000244529	32716
+Q	7	13	2	0.000305539	32729
+Q	6	47	1	0.000335611	32776
+Q	3	10	1	0.000366010	32786
+Q	2	20	2	0.000426751	32806
+Q	1	248	94	0.003267381	33054
+Q	0	31	17	0.003778147	33085

tex/roc.gp

@@ -14,7 +14,7 @@ set size 1.59,1.04
 set multiplot layout 1,2
 
 set label "(a)" at graph -0.245,1.06 font "Helvetica-bold,40"
-set xlab "Error rate of mapped reads"
+set xlab "Error rate of mapped PacBio reads"
 set ylab "Fraction of mapped reads" off +1.8
 set ytics 0.02
 set yran [0.9:1]
@@ -34,19 +34,27 @@ unset label
 set origin 0.8,0
 set size 0.79,1
 set label "(b)" at graph -0.245,1.06 font "Helvetica-bold,40"
-unset log
-unset format
-unset key
-set log y
-set ylab "Accumulative mapping error rate" off +0
-set xlab "Mapping quality"
-set yran [1e-5:0.1]
-set ytics 1e-5,0.1
-set format y "10^{%L}"
-set xran [60:0] reverse
-plot "<./eval2roc.pl blasr-mc.eval" u 1:2 w lp ls 4, \
-     "<./eval2roc.pl bwa.eval" u 1:2 t "bwa-mem" w lp ls 2, \
-     "<./eval2roc.pl graphmap.eval" u 1:2 t "graphmap" w lp ls 3, \
-     "<./eval2roc.pl minialign.eval" u 1:2 t "minialign" w lp ls 1, \
-     "<./eval2roc.pl mm2.eval" u 1:2 t "minimap2" w lp ls 6, \
-     "<./eval2roc.pl ngmlr.eval" u 1:2 t "ngm-lr" w lp ls 5
+set xlab "Error rate of mapped short reads"
+
+set key top left
+plot "<./eval2roc.pl -n2e7 bowtie2-s3.sam.eval" u 2:3 t "bowtie2" w lp ls 5, \
+	 "<./eval2roc.pl -n2e7 bwa-s3.sam.eval" u 2:3 t "bwa-mem" w lp ls 2, \
+	 "<./eval2roc.pl -n2e7 mm2-s3.sam.eval" u 2:3 t "minimap2" w lp ls 6, \
+	 "<./eval2roc.pl -n2e7 snap-s3.sam.eval" u 2:3 t "snap" w lp ls 3
+
+#unset log
+#unset format
+#unset key
+#set log y
+#set ylab "Accumulative mapping error rate" off +0
+#set xlab "Mapping quality"
+#set yran [1e-5:0.1]
+#set ytics 1e-5,0.1
+#set format y "10^{%L}"
+#set xran [60:0] reverse
+#plot "<./eval2roc.pl blasr-mc.eval" u 1:2 w lp ls 4, \
+#     "<./eval2roc.pl bwa.eval" u 1:2 t "bwa-mem" w lp ls 2, \
+#     "<./eval2roc.pl graphmap.eval" u 1:2 t "graphmap" w lp ls 3, \
+#     "<./eval2roc.pl minialign.eval" u 1:2 t "minialign" w lp ls 1, \
+#     "<./eval2roc.pl mm2.eval" u 1:2 t "minimap2" w lp ls 6, \
+#     "<./eval2roc.pl ngmlr.eval" u 1:2 t "ngm-lr" w lp ls 5

tex/snap-s3.sam.eval

@@ -0,0 +1,62 @@
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+U	8764