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200 lines
11 KiB
Markdown
200 lines
11 KiB
Markdown
## Getting Started
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```sh
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# Install hifiasm (requiring g++ and zlib)
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git clone https://github.com/chhylp123/hifiasm
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cd hifiasm && make
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# Run on test data (use -f0 for small datasets)
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wget https://github.com/chhylp123/hifiasm/releases/download/v0.7/chr11-2M.fa.gz
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./hifiasm -o test -t4 -f0 chr11-2M.fa.gz 2> test.log
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awk '/^S/{print ">"$2;print $3}' test.p_ctg.gfa > test.p_ctg.fa # get primary contigs in FASTA
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# Assemble inbred/homozygous genomes (-l0 disables duplication purging)
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hifiasm -o CHM13.asm -t32 -l0 CHM13-HiFi.fa.gz 2> CHM13.asm.log
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# Assemble heterozygous with built-in duplication purging
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hifiasm -o HG002.asm -t32 HG002-file1.fq.gz HG002-file2.fq.gz
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# Trio binning assembly (requiring https://github.com/lh3/yak)
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yak count -b37 -t16 -o pat.yak <(cat pat_1.fq.gz pat_2.fq.gz) <(cat pat_1.fq.gz pat_2.fq.gz)
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yak count -b37 -t16 -o mat.yak <(cat mat_1.fq.gz mat_2.fq.gz) <(cat mat_1.fq.gz mat_2.fq.gz)
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hifiasm -o HG002.asm -t32 -1 pat.yak -2 mat.yak HG002-HiFi.fa.gz
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```
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## Introduction
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Hifiasm is a fast haplotype-resolved de novo assembler for PacBio Hifi reads.
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It can assemble a human genome in several hours and works with the California
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redwood genome, one of the most complex genomes sequenced so far. Hifiasm can
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produce primary/alternate assemblies of quality competitive with the best
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assemblers. It also introduces a new graph binning algorithm and achieves
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the best haplotype-resolved assembly given trio data.
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## Why Hifiasm?
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* Hifiasm delivers high-quality assemblies. It tends to generate longer contigs
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and resolve more segmental duplications than other assemblers.
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* Given sequence reads from the parents, hifiasm can produce overall the best
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haplotype-resolved assembly so far. It is the assembler of choice by the
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[Human Pangenome Project][hpp] for the first batch of samples.
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* Hifiasm can purge duplications between haplotigs without relying on
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third-party tools such as purge\_dups. Hifiasm does not need polishing tools
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like pilon or racon, either. This simplifies the assembly pipeline and saves
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running time.
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* Hifiasm is fast. It can assemble a human genome in half a day and assemble a
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~30Gb redwood genome in three days. No genome is too large for hifiasm.
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* Hifiasm is trivial to install and easy to use. It does not required python,
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R or C++11 compilers and can be compiled into a single executable. The
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default setting works well with a variety of genomes.
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[hpp]: https://humanpangenome.org
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## Usage
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A typical hifiasm command line looks like:
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```sh
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hifiasm -o NA12878.asm -t 32 NA12878.fq.gz
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```
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where `NA12878.fq.gz` provides the input reads, `-t` sets the number of CPUs in
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use and `-o` specifies the prefix of output files. For this example, the
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primary contigs are written to `NA12878.asm.p_ctg.gfa` and alternate contigs to
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`NA12878.asm.a_ctg.gfa`. At the first run, hifiasm saves corrected reads and
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overlaps to disk as `NA12878.asm.*.bin`. It reuses the saved results to avoid
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the time-consuming all-vs-all overlap calculation next time. You may specify
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`-i` to ignore precomputed overlaps and redo overlapping from raw reads.
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Hifiasm purges haplotig duplications by default. For inbred or homozygous
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genomes, you may disable purging with option `-l0`. Old HiFi reads may contain
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short adapter sequences at the ends of reads. You can specify `-z20` to trim
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both ends of reads by 20bp. For small genomes, use `-f0` to disable the initial
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bloom filter which takes 16GB memory at the beginning. For genomes much larger
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than human, applying `-f38` or even `-f39` is preferred to save memory on k-mer
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counting.
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When parental short reads are available, hifiasm can generate a pair of
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haplotype-resolved assemblies with trio binning. To perform such assembly, you
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need to count k-mers first with [yak][yak] first and then do assembly:
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```sh
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yak count -k31 -b37 -t16 -o pat.yak paternal.fq.gz
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yak count -k31 -b37 -t16 -o mat.yak maternal.fq.gz
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hifiasm -o NA12878.asm -t 32 -1 pat.yak -2 mat.yak NA12878.fq.gz
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```
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Here `NA12878.asm.hap1.p_ctg.gfa` and `NA12878.asm.hap2.p_ctg.gfa` give the two
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haplotype assemblies. In the binning mode, hifiasm does not purge haplotig
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duplications by default. Because hifiasm reuses saved overlaps, you can
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generate both primary/alternate assemblies and trio binning assemblies with
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```sh
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hifiasm -o NA12878.asm -t 32 NA12878.fq.gz 2> NA12878.asm.pri.log
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hifiasm -o NA12878.asm -t 32 -1 pat.yak -2 mat.yak /dev/null 2> NA12878.asm.trio.log
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```
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The second command line will run much faster than the first. You can also dump
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error corrected in FASTA and/or overlaps in PAF with
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```sh
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hifiasm -o NA12878.asm -t 32 --write-paf --write-ec /dev/null
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```
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## Output files
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For non-trio assembly, hifiasm generates the following files:
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1. Haplotype-resolved raw [unitig][unitig] graph in [GFA][gfa] format
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(*prefix*.r\_utg.gfa). This graph keeps all haplotype information, including
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somatic mutations and recurrent sequencing errors.
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2. Haplotype-resolved processed unitig graph without small bubbles
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(*prefix*.p\_utg.gfa). Small bubbles might be caused by somatic mutations or noise in data,
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which are not the real haplotype information.
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3. Primary assembly [contig][unitig] graph (*prefix*.p\_ctg.gfa). This graph collapses different
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haplotypes.
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4. Alternate assembly contig graph (*prefix*.a\_ctg.gfa). This graph consists of all assemblies that
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are discarded in primary contig graph.
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For trio assembly, hifiasm generates the following files:
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1. Haplotype-resolved raw [unitig][unitig] graph in [GFA][gfa] format
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(*prefix*.r\_utg.gfa). This graph keeps all haplotype information.
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2. Phased paternal/haplotype1 contig graph (*prefix*.hap1.p\_ctg.gfa). This graph keeps the phased
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paternal/haplotype1 assembly.
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3. Phased maternal/haplotype2 contig graph (*prefix*.hap2.p\_ctg.gfa). This graph keeps the phased
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maternal/haplotype2 assembly.
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Hifiasm writes error corrected reads to the *prefix*.ec.bin binary file and
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writes overlaps to *prefix*.ovlp.source.bin and *prefix*.ovlp.reverse.bin.
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## Results
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The following table shows the statistics of several hifiasm primary assemblies:
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|<sub>Dataset<sub>|<sub>Size<sub>|<sub>Cov.<sub>|<sub>Asm options<sub>|<sub>CPU time<sub>|<sub>Wall time<sub>|<sub>RAM<sub>|<sub> N50<sub>|
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|:---------------|-----:|-----:|:---------------------|-------:|--------:|----:|----------------:|
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|<sub>[Mouse (C57/BL6J)][mouse-data]</sub>|<sub>2.6Gb</sub> |<sub>×25</sub>|<sub>-t48 -l0</sub> |<sub>172.9h</sub> |<sub>4.8h</sub> |<sub>76G</sub> |<sub>21.1Mb</sub>|
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|<sub>[Maize (B73)][maize-data]</sub> |<sub>2.2Gb</sub> |<sub>×22</sub>|<sub>-t48 -l0</sub> |<sub>203.2h</sub> |<sub>5.1h</sub> |<sub>68G</sub> |<sub>36.7Mb</sub>|
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|<sub>[Strawberry][strawberry-data]</sub> |<sub>0.8Gb</sub> |<sub>×36</sub>|<sub>-t48 -D10</sub>|<sub>152.7h</sub> |<sub>3.7h</sub> |<sub>91G</sub> |<sub>17.8Mb</sub>|
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|<sub>[Frog][frog-data]</sub> |<sub>9.5Gb</sub> |<sub>×29</sub>|<sub>-t48</sub> |<sub>2834.3h</sub>|<sub>69.0h</sub>|<sub>463G</sub>|<sub>9.3Mb</sub>|
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|<sub>[Redwood][redwood-data]</sub> |<sub>35.6Gb</sub>|<sub>×28</sub>|<sub>-t80</sub> |<sub>3890.3h</sub>|<sub>65.5h</sub>|<sub>699G</sub>|<sub>5.4Mb</sub>|
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|<sub>[Human (CHM13)][CHM13-data]</sub> |<sub>3.1Gb</sub> |<sub>×32</sub>|<sub>-t48 -l0</sub> |<sub>310.7h</sub> |<sub>8.2h</sub> |<sub>114G</sub>|<sub>88.9Mb</sub>|
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|<sub>[Human (HG00733)][HG00733-data]</sub>|<sub>3.1Gb</sub>|<sub>×33</sub>|<sub>-t48</sub> |<sub>269.1h</sub> |<sub>6.9h</sub> |<sub>135G</sub>|<sub>69.9Mb</sub>|
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|<sub>[Human (HG002)][NA24385-data]</sub> |<sub>3.1Gb</sub> |<sub>×36</sub>|<sub>-t48</sub> |<sub>305.4h</sub> |<sub>7.7h</sub> |<sub>137G</sub>|<sub>98.7Mb</sub>|
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[mouse-data]: https://www.ncbi.nlm.nih.gov/sra/?term=SRR11606870
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[maize-data]: https://www.ncbi.nlm.nih.gov/sra/?term=SRR11606869
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[strawberry-data]: https://www.ncbi.nlm.nih.gov/sra/?term=SRR11606867
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[frog-data]: https://www.ncbi.nlm.nih.gov/sra?term=(SRR11606868)%20OR%20SRR12048570
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[redwood-data]: https://www.ncbi.nlm.nih.gov/sra/?term=SRP251156
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[CHM13-data]: https://www.ncbi.nlm.nih.gov/sra?term=(((SRR11292120)%20OR%20SRR11292121)%20OR%20SRR11292122)%20OR%20SRR11292123
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Hifiasm can assemble a 3.1Gb human genome in several hours or a ~30Gb hexaploid
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redwood genome in a few days on a single machine. For trio binning assembly:
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|<sub>Dataset<sub>|<sub>Cov.<sub>|<sub>CPU time<sub>|<sub>Elapsed time<sub>|<sub>RAM<sub>|<sub> N50<sub>|
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|:---------------|-----:|-------:|--------:|----:|----------------:|
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|<sub>[HG00733][HG00733-data], [\[father\]][HG00731-data], [\[mother\]][HG00732-data]</sub>|<sub>×33</sub>|<sub>269.1h</sub>|<sub>6.9h</sub>|<sub>135G</sub>|<sub>35.1Mb (paternal), 34.9Mb (maternal)</sub>|
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|<sub>[HG002][NA24385-data], [\[father\]][NA24149-data], [\[mother\]][NA24143-data]</sup>|<sub>×36</sub>|<sub>305.4h</sub>|<sub>7.7h</sub>|<sub>137G</sub>|<sub>41.0Mb (paternal), 40.8Mb (maternal)</sub>|
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|<sub>[NA12878][NA12878-data], [\[father\]][NA12891-data], [\[mother\]][NA12892-data]</sub>|<sub>×30</sub>|<sub>180.8h</sub>|<sub>4.9h</sub>|<sub>123G</sub>|<sub>27.7Mb (paternal), 27.0Mb (maternal)</sub>|
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[HG00733-data]: https://www.ebi.ac.uk/ena/data/view/ERX3831682
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[HG00731-data]: https://www.ebi.ac.uk/ena/data/view/ERR3241754
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[HG00732-data]: https://www.ebi.ac.uk/ena/data/view/ERR3241755
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[NA24385-data]: https://www.ncbi.nlm.nih.gov/sra?term=(((SRR10382244)%20OR%20SRR10382245)%20OR%20SRR10382248)%20OR%20SRR10382249
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[NA24149-data]: https://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/HG003_NA24149_father/NIST_HiSeq_HG003_Homogeneity-12389378/HG003Run01-13262252/
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[NA24143-data]: https://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/HG004_NA24143_mother/NIST_HiSeq_HG004_Homogeneity-14572558/HG004Run01-15133132/
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[NA12878-data]: https://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/NA12878/PacBio_SequelII_CCS_11kb/
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[NA12891-data]: https://www.ebi.ac.uk/ena/data/view/ERR194160
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[NA12892-data]: https://www.ebi.ac.uk/ena/data/view/ERR194161
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Except NA12878, the assemblies above were produced by hifiasm v0.12 and can be
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downloaded at
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```txt
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ftp://ftp.dfci.harvard.edu/pub/hli/hifiasm/submission/hifiasm-0.12/
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```
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NA12878 was assembled with an older version of hifiasm and is available at
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```txt
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ftp://ftp.dfci.harvard.edu/pub/hli/hifiasm/NA12878-r253/
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```
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[unitig]: http://wgs-assembler.sourceforge.net/wiki/index.php/Celera_Assembler_Terminology
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[gfa]: https://github.com/pmelsted/GFA-spec/blob/master/GFA-spec.md
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[paf]: https://github.com/lh3/miniasm/blob/master/PAF.md
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[yak]: https://github.com/lh3/yak
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## Getting Help
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For detailed description of options, please see `man ./hifiasm.1`. The `-h`
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option of hifiasm also provides brief description of options. If you have
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further questions, please raise an issue at the [issue
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page](https://github.com/chhylp123/hifiasm/issues).
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## Limitations
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1. Purging haplotig duplications may introduce misassemblies.
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## Citation
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Cheng, H., Concepcion, G.T., Feng, X., Zhang, H., Li H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods 18, 170–175 (2021). https://doi.org/10.1038/s41592-020-01056-5
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