190 lines
9.2 KiB
Markdown
190 lines
9.2 KiB
Markdown
[](https://github.com/lh3/bwa/actions)
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[](https://sourceforge.net/projects/bio-bwa/files/?source=navbar)
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[](https://github.com/lh3/bwa/releases)
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[](https://anaconda.org/bioconda/bwa)
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**Note: [minimap2][minimap2] has replaced BWA-MEM for __PacBio and Nanopore__ read
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alignment.** It retains all major BWA-MEM features, but is ~50 times as fast,
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more versatile, more accurate and produces better base-level alignment.
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A beta version of [BWA-MEM2][bwa-mem2] has been released for short-read mapping.
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BWA-MEM2 is about twice as fast as BWA-MEM and outputs near identical alignments.
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[minimap2]: https://github.com/lh3/minimap2
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[bwa-mem2]: https://github.com/bwa-mem2/bwa-mem2
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## Getting started
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git clone https://github.com/lh3/bwa.git
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cd bwa; make
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./bwa index ref.fa
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./bwa mem ref.fa read-se.fq.gz | gzip -3 > aln-se.sam.gz
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./bwa mem ref.fa read1.fq read2.fq | gzip -3 > aln-pe.sam.gz
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## Introduction
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BWA is a software package for mapping DNA sequences against a large reference
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genome, such as the human genome. It consists of three algorithms:
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BWA-backtrack, BWA-SW and BWA-MEM. The first algorithm is designed for Illumina
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sequence reads up to 100bp, while the rest two for longer sequences ranged from
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70bp to a few megabases. BWA-MEM and BWA-SW share similar features such as the
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support of long reads and chimeric alignment, but BWA-MEM, which is the latest,
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is generally recommended as it is faster and more accurate. BWA-MEM also has
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better performance than BWA-backtrack for 70-100bp Illumina reads.
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For all the algorithms, BWA first needs to construct the FM-index for the
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reference genome (the **index** command). Alignment algorithms are invoked with
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different sub-commands: **aln/samse/sampe** for BWA-backtrack,
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**bwasw** for BWA-SW and **mem** for the BWA-MEM algorithm.
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## Availability
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BWA is released under [GPLv3][1]. The latest source code is [freely
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available at github][2]. Released packages can [be downloaded][3] at
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SourceForge. After you acquire the source code, simply use `make` to compile
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and copy the single executable `bwa` to the destination you want. The only
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dependency required to build BWA is [zlib][14].
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Since 0.7.11, precompiled binary for x86\_64-linux is available in [bwakit][17].
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In addition to BWA, this self-consistent package also comes with bwa-associated
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and 3rd-party tools for proper BAM-to-FASTQ conversion, mapping to ALT contigs,
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adapter triming, duplicate marking, HLA typing and associated data files.
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## Seeking help
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The detailed usage is described in the man page available together with the
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source code. You can use `man ./bwa.1` to view the man page in a terminal. The
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[HTML version][4] of the man page can be found at the [BWA website][5]. If you
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have questions about BWA, you may [sign up the mailing list][6] and then send
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the questions to [bio-bwa-help@sourceforge.net][7]. You may also ask questions
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in forums such as [BioStar][8] and [SEQanswers][9].
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## Citing BWA
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* Li H. and Durbin R. (2009) Fast and accurate short read alignment with
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Burrows-Wheeler transform. *Bioinformatics*, **25**, 1754-1760. [PMID:
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[19451168][10]]. (if you use the BWA-backtrack algorithm)
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* Li H. and Durbin R. (2010) Fast and accurate long-read alignment with
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Burrows-Wheeler transform. *Bioinformatics*, **26**, 589-595. [PMID:
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[20080505][11]]. (if you use the BWA-SW algorithm)
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* Li H. (2013) Aligning sequence reads, clone sequences and assembly contigs
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with BWA-MEM. [arXiv:1303.3997v2][12] [q-bio.GN]. (if you use the BWA-MEM
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algorithm or the **fastmap** command, or want to cite the whole BWA package)
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Please note that the last reference is a preprint hosted at [arXiv.org][13]. I
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do not have plan to submit it to a peer-reviewed journal in the near future.
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## Frequently asked questions (FAQs)
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1. [What types of data does BWA work with?](#type)
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2. [Why does a read appear multiple times in the output SAM?](#multihit)
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3. [Does BWA work on reference sequences longer than 4GB in total?](#4gb)
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4. [Why can one read in a pair has high mapping quality but the other has zero?](#pe0)
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5. [How can a BWA-backtrack alignment stands out of the end of a chromosome?](#endref)
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6. [Does BWA work with ALT contigs in the GRCh38 release?](#altctg)
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7. [Can I just run BWA-MEM against GRCh38+ALT without post-processing?](#postalt)
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#### <a name="type"></a>1. What types of data does BWA work with?
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BWA works with a variety types of DNA sequence data, though the optimal
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algorithm and setting may vary. The following list gives the recommended
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settings:
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* Illumina/454/IonTorrent single-end reads longer than ~70bp or assembly
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contigs up to a few megabases mapped to a closely related reference genome:
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bwa mem ref.fa reads.fq > aln.sam
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* Illumina single-end reads shorter than ~70bp:
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bwa aln ref.fa reads.fq > reads.sai; bwa samse ref.fa reads.sai reads.fq > aln-se.sam
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* Illumina/454/IonTorrent paired-end reads longer than ~70bp:
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bwa mem ref.fa read1.fq read2.fq > aln-pe.sam
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* Illumina paired-end reads shorter than ~70bp:
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bwa aln ref.fa read1.fq > read1.sai; bwa aln ref.fa read2.fq > read2.sai
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bwa sampe ref.fa read1.sai read2.sai read1.fq read2.fq > aln-pe.sam
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* PacBio subreads or Oxford Nanopore reads to a reference genome:
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bwa mem -x pacbio ref.fa reads.fq > aln.sam
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bwa mem -x ont2d ref.fa reads.fq > aln.sam
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BWA-MEM is recommended for query sequences longer than ~70bp for a variety of
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error rates (or sequence divergence). Generally, BWA-MEM is more tolerant with
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errors given longer query sequences as the chance of missing all seeds is small.
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As is shown above, with non-default settings, BWA-MEM works with Oxford Nanopore
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reads with a sequencing error rate over 20%.
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#### <a name="multihit"></a>2. Why does a read appear multiple times in the output SAM?
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BWA-SW and BWA-MEM perform local alignments. If there is a translocation, a gene
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fusion or a long deletion, a read bridging the break point may have two hits,
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occupying two lines in the SAM output. With the default setting of BWA-MEM, one
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and only one line is primary and is soft clipped; other lines are tagged with
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0x800 SAM flag (supplementary alignment) and are hard clipped.
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#### <a name="4gb"></a>3. Does BWA work on reference sequences longer than 4GB in total?
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Yes. Since 0.6.x, all BWA algorithms work with a genome with total length over
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4GB. However, individual chromosome should not be longer than 2GB.
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#### <a name="pe0"></a>4. Why can one read in a pair have a high mapping quality but the other has zero?
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This is correct. Mapping quality is assigned for individual read, not for a read
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pair. It is possible that one read can be mapped unambiguously, but its mate
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falls in a tandem repeat and thus its accurate position cannot be determined.
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#### <a name="endref"></a>5. How can a BWA-backtrack alignment stand out of the end of a chromosome?
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Internally BWA concatenates all reference sequences into one long sequence. A
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read may be mapped to the junction of two adjacent reference sequences. In this
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case, BWA-backtrack will flag the read as unmapped (0x4), but you will see
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position, CIGAR and all the tags. A similar issue may occur to BWA-SW alignment
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as well. BWA-MEM does not have this problem.
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#### <a name="altctg"></a>6. Does BWA work with ALT contigs in the GRCh38 release?
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Yes, since 0.7.11, BWA-MEM officially supports mapping to GRCh38+ALT.
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BWA-backtrack and BWA-SW don't properly support ALT mapping as of now. Please
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see [README-alt.md][18] for details. Briefly, it is recommended to use
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[bwakit][17], the binary release of BWA, for generating the reference genome
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and for mapping.
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#### <a name="postalt"></a>7. Can I just run BWA-MEM against GRCh38+ALT without post-processing?
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If you are not interested in hits to ALT contigs, it is okay to run BWA-MEM
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without post-processing. The alignments produced this way are very close to
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alignments against GRCh38 without ALT contigs. Nonetheless, applying
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post-processing helps to reduce false mappings caused by reads from the
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diverged part of ALT contigs and also enables HLA typing. It is recommended to
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run the post-processing script.
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[1]: http://en.wikipedia.org/wiki/GNU_General_Public_License
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[2]: https://github.com/lh3/bwa
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[3]: http://sourceforge.net/projects/bio-bwa/files/
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[4]: http://bio-bwa.sourceforge.net/bwa.shtml
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[5]: http://bio-bwa.sourceforge.net/
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[6]: https://lists.sourceforge.net/lists/listinfo/bio-bwa-help
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[7]: mailto:bio-bwa-help@sourceforge.net
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[8]: http://biostars.org
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[9]: http://seqanswers.com/
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[10]: http://www.ncbi.nlm.nih.gov/pubmed/19451168
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[11]: http://www.ncbi.nlm.nih.gov/pubmed/20080505
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[12]: http://arxiv.org/abs/1303.3997
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[13]: http://arxiv.org/
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[14]: http://zlib.net/
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[15]: https://github.com/lh3/bwa/tree/mem
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[16]: ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Homo_sapiens/GRCh38/seqs_for_alignment_pipelines/
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[17]: http://sourceforge.net/projects/bio-bwa/files/bwakit/
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[18]: https://github.com/lh3/bwa/blob/master/README-alt.md
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# fast-bwa
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基于bwa，做一些优化，注释
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