rpique / bwa

Burrow-Wheeler Aligner

##Getting started

git clone https://github.com/lh3/bwa.git
cd bwa; make
./bwa index ref.fa
./bwa mem ref.fa read-se.fq.gz | gzip -3 > aln-se.sam.gz
./bwa mem ref.fa read1.fq read2.fq | gzip -3 > aln-pe.sam.gz

##Introduction

BWA is a software package for mapping DNA sequences against a large reference genome, such as the human genome. It consists of three algorithms: BWA-backtrack, BWA-SW and BWA-MEM. The first algorithm is designed for Illumina sequence reads up to 100bp, while the rest two for longer sequences ranged from 70bp to a few megabases. BWA-MEM and BWA-SW share similar features such as the support of long reads and chimeric alignment, but BWA-MEM, which is the latest, is generally recommended as it is faster and more accurate. BWA-MEM also has better performance than BWA-backtrack for 70-100bp Illumina reads.

For all the algorithms, BWA first needs to construct the FM-index for the reference genome (the index command). Alignment algorithms are invoked with different sub-commands: aln/samse/sampe for BWA-backtrack, bwasw for BWA-SW and mem for the BWA-MEM algorithm.

##Availability

BWA is released under GPLv3. The latest source code is freely available at github. Released packages can be downloaded at SourceForge. After you acquire the source code, simply use make to compile and copy the single executable bwa to the destination you want. The only dependency required to build BWA is zlib.

##Seeking helps

The detailed usage is described in the man page available together with the source code. You can use man ./bwa.1 to view the man page in a terminal. The HTML version of the man page can be found at the BWA website. If you have questions about BWA, you may sign up the mailing list and then send the questions to [email protected]. You may also ask questions in forums such as BioStar and SEQanswers.

##Citing BWA

• Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25, 1754-1760. [PMID: 19451168]. (if you use the BWA-backtrack algorithm)

• Li H. and Durbin R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics, 26, 589-595. [PMID: 20080505]. (if you use the BWA-SW algorithm)

• Li H. (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv:1303.3997v2 [q-bio.GN]. (if you use the BWA-MEM algorithm or the fastmap command, or want to cite the whole BWA package)

Please note that the last reference is a preprint hosted at arXiv.org. I do not have plan to submit it to a peer-reviewed journal in the near future.

##Frequently asked questions (FAQs)

1. What types of data does BWA work with?
2. Why does a read appear multiple times in the output SAM?
3. Does BWA work on reference sequences longer than 4GB in total?
4. Why can one read in a pair has high mapping quality but the other has zero?
5. How can a BWA-backtrack alignment stands out of the end of a chromosome?
6. How to map sequences to GRCh38 with ALT contigs?

####1. What types of data does BWA work with?

BWA works with a variety types of DNA sequence data, though the optimal algorithm and setting may vary. The following list gives the recommended settings:

• Illumina/454/IonTorrent single-end reads longer than ~70bp or assembly contigs up to a few megabases mapped to a close related reference genome:

    bwa mem ref.fa reads.fq > aln.sam
  

• Illumina single-end reads no longer than ~70bp:

    bwa aln ref.fa reads.fq > reads.sai; bwa samse ref.fa reads.sai reads.fq > aln-se.sam
  

• Illumina/454/IonTorrent paired-end reads longer than ~70bp:

    bwa mem ref.fa read1.fq read2.fq > aln-pe.sam
  

• Illumina paired-end reads no longer than ~70bp:

    bwa aln ref.fa read1.fq > read1.sai; bwa aln ref.fa read2.fq > read2.sai
    bwa samse ref.fa reads.sai reads.fq > aln-pe.sam
  

• PacBio subreads to a reference genome:

    bwa mem -x pacbio ref.fa reads.fq > aln.sam
  

• PacBio subreads to themselves (the output is not SAM):

    bwa mem -x pbread reads.fq reads.fq > overlap.pas
  

BWA-MEM is recommended for query sequences longer than ~70bp for a variety of error rates (or sequence divergence). Generally, BWA-MEM is more tolerant with errors given longer query sequences as the chance of missing all seeds is small. As is shown above, with non-default settings, BWA-MEM works with PacBio subreads with a sequencing error rate as high as ~15%.

####2. Why does a read appear multiple times in the output SAM?

BWA-SW and BWA-MEM perform local alignments. If there is a translocation, a gene fusion or a long deletion, a read bridging the break point may have two hits, occupying two lines in the SAM output. With the default setting of BWA-MEM, one and only one line is primary and is soft clipped; other lines are tagged with 0x800 SAM flag (supplementary alignment) and are hard clipped.

####3. Does BWA work on reference sequences longer than 4GB in total?

Yes. Since 0.6.x, all BWA algorithms work with a genome with total length over 4GB. However, individual chromosome should not be longer than 2GB.

####4. Why can one read in a pair has high mapping quality but the other has zero?

This is correct. Mapping quality is assigned for individual read, not for a read pair. It is possible that one read can be mapped unambiguously, but its mate falls in a tandem repeat and thus its accurate position cannot be determined.

####5. How can a BWA-backtrack alignment stands out of the end of a chromosome?

Internally BWA concatenates all reference sequences into one long sequence. A read may be mapped to the junction of two adjacent reference sequences. In this case, BWA-backtrack will flag the read as unmapped (0x4), but you will see position, CIGAR and all the tags. A similar issue may occur to BWA-SW alignment as well. BWA-MEM does not have this problem.

####6. How to map sequences to GRCh38 with ALT contigs?

BWA-backtrack and BWA-MEM partially support mapping to a reference containing ALT contigs that represent alternative alleles highly divergent from the reference genome.

# download the K8 executable required by bwa-helper.js
wget http://sourceforge.net/projects/lh3/files/k8/k8-0.2.1.tar.bz2/download
tar -jxf k8-0.2.1.tar.bz2

# download the ALT-to-GRCh38 alignment in the SAM format
wget http://sourceforge.net/projects/bio-bwa/files/hs38.alt.sam.gz/download

# download the GRCh38 sequences with ALT contigs
wget ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Homo_sapiens/GRCh38/seqs_for_alignment_pipelines/GCA_000001405.15_GRCh38_full_analysis_set.fna.gz

# index and mapping
bwa index -p hs38a GCA_000001405.15_GRCh38_full_analysis_set.fna.gz
bwa mem -h50 hs38a reads.fq | ./k8-linux bwa-helper.js genalt hs38.alt.sam.gz > out.sam

Here, option -h50 asks bwa-mem to output multiple hits in the XA tag if the read has 50 or fewer hits. For each SAM line containing the XA tag, bwa-helper.js genalt decodes the alignments in the XA tag, groups hits lifted to the same chromosomal region, adjusts mapping quality and outputs all the hits overlapping the reported hit. A read may be mapped to both the primary assembly and one or more ALT contigs all with high mapping quality.

Note that this procedure assumes reads are single-end and may miss hits to highly repetitive regions as these hits will not be reported with option -h50. bwa-helper.js is a prototype implementation not recommended for production uses.