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Heng Li
2020-06-27 19:25:38 -04:00
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README.md
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@@ -7,10 +7,11 @@ cd hifiasm && make
# Run on test data (use -f0 for small datasets)
wget https://github.com/chhylp123/hifiasm/releases/download/v0.7/chr11-2M.fa.gz
./hifiasm -o test -t4 -f0 chr11-2M.fa.gz 2> test.log # this takes ~90 sec
./hifiasm -o test -t4 -f0 chr11-2M.fa.gz 2> test.log
awk '/^S/{print ">"$1;print $2}' test.p_ctg.gfa > test.p_ctg.fa # get primary contigs in FASTA
# Assemble inbred/homozygous genomes (-l0 disables duplication purging)
hifiasm -o CHM13.asm -t32 -l0 CHM13-HiFi.fa.gz
hifiasm -o CHM13.asm -t32 -l0 CHM13-HiFi.fa.gz 2> CHM13.asm.log
# Assemble heterozygous with built-in duplication purging
hifiasm -o HG002.asm -t32 HG002-file1.fq.gz HG002-file2.fq.gz
@@ -22,62 +23,60 @@ hifiasm -o HG002.asm -t32 -1 pat.yak -2 mat.yak HG002-HiFi.fa.gz
## Introduction
Hifiasm is a fast haplotype-resolved de novo assembler for PacBio
Hifi reads. Unlike most existing assemblers, hifiasm starts from uncollapsed
genome. Thus, it is able to keep the haplotype information as much as possible.
Hifiasm supports both primary assembly and fully phased assembly (trio-binning assembly).
Hifiasm is a fast haplotype-resolved de novo assembler for PacBio Hifi reads.
It can assemble a human genome in several hours and works with the California
redwood genome, one of the most complex genomes sequenced so far. Hifiasm can
produce primary/alternate assemblies of quality competitive with the best
assemblers. It also introduces a new graph binning algorithm and achieves
the best haplotype-resolved assembly given trio data.
## Usage
For Hifi reads assembly, a typical command line looks like:
A typical hifiasm command line looks like:
```sh
./hifiasm -o NA12878.asm -t 32 NA12878.fq.gz
hifiasm -o NA12878.asm -t 32 NA12878.fq.gz
```
where `NA12878.fq.gz` provides the input reads, `-t` sets the number of CPUs in
use and `-o` specifies the prefix of output files. For this example, the
primary contigs are written to `NA12878.asm.p_ctg.gfa` and alternate contigs to
`NA12878.asm.a_ctg.gfa`. At the first run, hifiasm saves corrected reads and
overlaps to disk as `NA12878.asm.*.bin`. It reuses the saved results to avoid
the time-consuming all-vs-all overlap calculation next time. You may specify
`-i` to ignore precomputed overlaps and redo overlapping from raw reads.
Hifiasm purges haplotig duplications by default. For inbred or homozygous
genomes, you may disable purging with option `-l0`. Old HiFi reads may contain
short adapter sequences at the ends of reads. You can specify `-z20` to trim
both ends of reads by 20bp. For small genomes, use `-f0` to disable the initial
bloom filter which takes 16GB memory at the beginning. For genomes much larger
than human, applying `-f38` or even `-f39` is preferred to save memory on k-mer
counting.
When parental short reads are available, hifiasm can generate a pair of
haplotype-resolved assemblies with trio binning. To perform such assembly, you
need to count k-mers first with [yak][yak] first and then do assembly:
```sh
yak count -k31 -b37 -t16 -o pat.yak paternal.fq.gz
yak count -k31 -b37 -t16 -o mat.yak maternal.fq.gz
hifiasm -o NA12878.asm -t 32 -1 pat.yak -2 mat.yak NA12878.fq.gz
```
Here `NA12878.asm.hap1.p_ctg.gfa` and `NA12878.asm.hap2.p_ctg.gfa` give the two
haplotype assemblies. In the binning mode, hifiasm does not purge haplotig
duplications by default. Because hifiasm reuses saved overlaps, you can
generate both primary/alternate assemblies and trio binning assemblies with
```sh
hifiasm -o NA12878.asm -t 32 NA12878.fq.gz 2> NA12878.asm.pri.log
hifiasm -o NA12878.asm -t 32 -1 pat.yak -2 mat.yak /dev/null 2> NA12878.asm.trio.log
```
The second command line will run much faster than the first. You can also dump
error corrected in FASTA and/or overlaps in PAF with
```sh
hifiasm -o NA12878.asm -t 32 --write-paf --write-ec /dev/null
```
where `NA12878.fq.gz` is the input reads and `-o` specifies the output files.
In this example, all output files can be found at `NA12878.asm.*`. `-t` specifies
the number of CPU threads. Note that at first run, hifiasm will save all overlaps
to disk, which can avoid the time-consuming all-to-all overlap calculation next time.
For hifiasm, once the overlap information has been obtained during the previous run
in advance, it is able to load all overlaps from disk and then directly do assembly.
If you want to ignore the pre-computed overlap information, please specify `-i`.
The example dataset can be found at: https://github.com/chhylp123/hifiasm/releases/download/v0.7/chr11-2M.fa.gz.
## Output files
Please note that some old Hifi reads may consist of short adapters. To improve
the assembly quality, adapters should be removed by `-z` as follow:
```sh
./hifiasm -o butterfly.asm -t 42 -z 20 butterfly.fq.gz
```
In this example, hifiasm will remove 20 bases from both ends of each read.
For trio assembly, first the trio indexes of paternal/maternal should be generated by
[yak count](https://github.com/lh3/yak):
```sh
./yak count -k31 -b37 -t16 -o mat.yak mat.fq.gz
./yak count -k31 -b37 -t16 -o pat.yak pat.fq.gz
```
and then run hifiasm as follow:
```sh
./hifiasm -o NA12878.asm -t 32 -1 pat.yak -2 mat.yak NA12878_1.fq.gz NA12878_2.fq.gz
```
## Advance features
For primary assembly, hifiasm performs purge duplication step in default. This step is designed for diploid genomes or polyploid genomes to keep one set of haplotypes without duplications. For inbred genomes or homozygous samples including only one haplotype, purge duplication step may introduce misassemblies so that it should be disabled by option `-l0`.
## Output
For non-trio assembly, the input of hifiasm is the PacBio Hifi reads in fasta/fastq format, and its
outputs consist of:
For non-trio assembly, hifiasm generates the following files:
1. Haplotype-resolved raw [unitig][unitig] graph in [GFA][gfa] format
(*prefix*.r\_utg.gfa). This graph keeps all haplotype information, including
@@ -90,7 +89,8 @@ outputs consist of:
4. Alternate assembly contig graph (*prefix*.a\_ctg.gfa). This graph consists of all assemblies that
are discarded in primary contig graph.
For trio assembly, the input of hifiasm is the PacBio Hifi reads in fasta/fastq format, and the paternal/maternal trio indexes generated by [yak count](https://github.com/lh3/yak). The outputs consist of:
For trio assembly, hifiasm generates the following files:
1. Haplotype-resolved raw [unitig][unitig] graph in [GFA][gfa] format
(*prefix*.r\_utg.gfa). This graph keeps all haplotype information.
@@ -100,17 +100,12 @@ For trio assembly, the input of hifiasm is the PacBio Hifi reads in fasta/fastq
3. Phased maternal/haplotype2 contig graph (*prefix*.hap2.p\_ctg.gfa). This graph keeps the phased
maternal/haplotype2 assembly.
In addition, hifiasm also outputs three binary files that save all overlap information (*prefix*.ec.bin, *prefix*.ovlp.reverse.bin, *prefix*.ovlp.source.bin). With these files, hifiasm can avoid the time-consuming all-to-all overlap calculation step, and do the assembly
directly and quickly. This might be helpful when you want to get an optimized
assembly by multiple rounds of experiments with different parameters.
Hifiasm writes error corrected reads to the *prefix*.ec.bin binary file and
writes overlaps to *prefix*.ovlp.source.bin and *prefix*.ovlp.reverse.bin.
## Results
Hifiasm is a standalone and lightweight assembler, which does not need external
libraries (except zlib). For large genomes, it can generate high-quality primary
assembly in several hours. Hifiasm has been tested on various large and complex datasets.
The results are as follows:
The following table shows the statistics of several hifiasm primary assemblies:
|<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>|
|:---------------|-----:|-----:|:---------------------|-------:|--------:|----:|----------------:|
@@ -130,7 +125,9 @@ The results are as follows:
[redwood-data]: https://www.ncbi.nlm.nih.gov/sra/?term=SRP251156
[CHM13-data]: https://www.ncbi.nlm.nih.gov/sra?term=(((SRR11292120)%20OR%20SRR11292121)%20OR%20SRR11292122)%20OR%20SRR11292123
Hifiasm also can produce high-quality fully phased assembly. We tested it on the following trio-binning datasets:
Hifiasm can assemble a 3.1Gb human genome in several hours or a ~30Gb hexaploid
redwood genome in a few days on a single machine. For trio binning assembly:
|<sub>Dataset<sub>|<sub>Cov.<sub>|<sub>CPU time<sub>|<sub>Elapsed time<sub>|<sub>RAM<sub>|<sub> N50<sub>|
|:---------------|-----:|-------:|--------:|----:|----------------:|
|<sub>[HG00733][HG00733-data], [\[father\]][HG00731-data], [\[mother\]][HG00732-data]</sub>|<sub>&times;33</sub>|<sub>269.1h</sub>|<sub>6.9h</sub>|<sub>135G</sub>|<sub>35.1Mb (paternal), 34.9Mb (maternal)</sub>|
@@ -161,15 +158,15 @@ ftp://ftp.dfci.harvard.edu/pub/hli/hifiasm/NA12878-r253/
[unitig]: http://wgs-assembler.sourceforge.net/wiki/index.php/Celera_Assembler_Terminology
[gfa]: https://github.com/pmelsted/GFA-spec/blob/master/GFA-spec.md
[paf]: https://github.com/lh3/miniasm/blob/master/PAF.md
[yak]: https://github.com/lh3/yak
## Getting Help
For detailed description of options, please see `man ./hifiasm.1`.
The `-h` option of hifiasm also provides simple description of options. If you
have further questions, please raise an issue at the issue page.
For detailed description of options, please see `man ./hifiasm.1`. The `-h`
option of hifiasm also provides brief description of options. If you have
further questions, please raise an issue at the [issue
page](https://github.com/chhylp123/hifiasm/issues).
## Limitations and future works
## Limitations
1. The running time and memory usage should be further reduced.
2. The N50 should be further improved.
1. Purging haplotig duplications may introduce misassemblies.