chhylp123 / Hifiasm
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Getting Started
# Install hifiasm (requiring g++ and zlib)
git clone https://github.com/chhylp123/hifiasm
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
awk '/^S/{print ">"$2;print $3}' 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 2> CHM13.asm.log
# Assemble heterozygous with built-in duplication purging
hifiasm -o HG002.asm -t32 HG002-file1.fq.gz HG002-file2.fq.gz
# Trio binning assembly (requiring https://github.com/lh3/yak)
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)
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)
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. 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.
Why Hifiasm?
-
Hifiasm delivers high-quality assemblies. It tends to generate longer contigs and resolve more segmental duplications than other assemblers.
-
Given sequence reads from the parents, hifiasm can produce overall the best haplotype-resolved assembly so far. It is the assembler of choice by the Human Pangenome Project for the first batch of samples.
-
Hifiasm can purge duplications between haplotigs without relying on third-party tools such as purge_dups. Hifiasm does not need polishing tools like pilon or racon, either. This simplifies the assembly pipeline and saves running time.
-
Hifiasm is fast. It can assemble a human genome in half a day and assemble a ~30Gb redwood genome in three days. No genome is too large for hifiasm.
-
Hifiasm is trivial to install and easy to use. It does not required python, R or C++11 compilers and can be compiled into a single executable. The default setting works well with a variety of genomes.
Usage
A typical hifiasm command line looks like:
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 first and then do assembly:
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
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
hifiasm -o NA12878.asm -t 32 --write-paf --write-ec /dev/null
Output files
For non-trio assembly, hifiasm generates the following files:
- Haplotype-resolved raw unitig graph in GFA format (prefix.r_utg.gfa). This graph keeps all haplotype information, including somatic mutations and recurrent sequencing errors.
- Haplotype-resolved processed unitig graph without small bubbles (prefix.p_utg.gfa). Small bubbles might be caused by somatic mutations or noise in data, which are not the real haplotype information.
- Primary assembly contig graph (prefix.p_ctg.gfa). This graph collapses different haplotypes.
- Alternate assembly contig graph (prefix.a_ctg.gfa). This graph consists of all assemblies that are discarded in primary contig graph.
For trio assembly, hifiasm generates the following files:
-
Haplotype-resolved raw unitig graph in GFA format (prefix.r_utg.gfa). This graph keeps all haplotype information.
-
Phased paternal/haplotype1 contig graph (prefix.hap1.p_ctg.gfa). This graph keeps the phased paternal/haplotype1 assembly.
-
Phased maternal/haplotype2 contig graph (prefix.hap2.p_ctg.gfa). This graph keeps the phased maternal/haplotype2 assembly.
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
The following table shows the statistics of several hifiasm primary assemblies:
Dataset | Size | Cov. | Asm options | CPU time | Wall time | RAM | N50 |
---|---|---|---|---|---|---|---|
Mouse (C57/BL6J) | 2.6Gb | ×25 | -t48 -l0 | 172.9h | 4.8h | 76G | 21.1Mb |
Maize (B73) | 2.2Gb | ×22 | -t48 -l0 | 203.2h | 5.1h | 68G | 36.7Mb |
Strawberry | 0.8Gb | ×36 | -t48 -D10 | 152.7h | 3.7h | 91G | 17.8Mb |
Frog | 9.5Gb | ×29 | -t48 | 2834.3h | 69.0h | 463G | 9.3Mb |
Redwood | 35.6Gb | ×28 | -t80 | 3890.3h | 65.5h | 699G | 5.4Mb |
Human (CHM13) | 3.1Gb | ×32 | -t48 -l0 | 310.7h | 8.2h | 114G | 88.9Mb |
Human (HG00733) | 3.1Gb | ×33 | -t48 | 269.1h | 6.9h | 135G | 69.9Mb |
Human (HG002) | 3.1Gb | ×36 | -t48 | 305.4h | 7.7h | 137G | 98.7Mb |
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:
Dataset | Cov. | CPU time | Elapsed time | RAM | N50 |
---|---|---|---|---|---|
HG00733, [father], [mother] | ×33 | 269.1h | 6.9h | 135G | 35.1Mb (paternal), 34.9Mb (maternal) |
HG002, [father], [mother] | ×36 | 305.4h | 7.7h | 137G | 41.0Mb (paternal), 40.8Mb (maternal) |
NA12878, [father], [mother] | ×30 | 180.8h | 4.9h | 123G | 27.7Mb (paternal), 27.0Mb (maternal) |
Except NA12878, the assemblies above were produced by hifiasm v0.12 and can be downloaded at
ftp://ftp.dfci.harvard.edu/pub/hli/hifiasm/submission/hifiasm-0.12/
NA12878 was assembled with an older version of hifiasm and is available at
ftp://ftp.dfci.harvard.edu/pub/hli/hifiasm/NA12878-r253/
Getting Help
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.
Limitations
- Purging haplotig duplications may introduce misassemblies.
Citation
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