NAME
bwa - Burrows-Wheeler Alignment Tool
SYNOPSIS
bwa index -a bwtsw database.fasta
bwa aln database.fasta short_read.fastq > aln_sa.sai
bwa samse database.fasta aln_sa.sai short_read.fastq > aln.sam
bwa sampe database.fasta aln_sa1.sai aln_sa2.sai read1.fq read2.fq >
aln.sam
bwa bwasw database.fasta long_read.fastq > aln.sam
DESCRIPTION
BWA is a fast light-weighted tool that aligns relatively short
sequences (queries) to a sequence database (targe), such as the human
reference genome. It implements two different algorithms, both based on
Burrows-Wheeler Transform (BWT). The first algorithm is designed for
short queries up to ~200bp with low error rate (<3%). It does gapped
global alignment w.r.t. queries, supports paired-end reads, and is one
of the fastest short read alignment algorithms to date while also
visiting suboptimal hits. The second algorithm, BWA-SW, is designed for
long reads with more errors. It performs heuristic Smith-Waterman-like
alignment to find high-scoring local hits (and thus chimera). On low-
error short queries, BWA-SW is slower and less accurate than the first
algorithm, but on long queries, it is better.
For both algorithms, the database file in the FASTA format must be
first indexed with the ‘index’ command, which typically takes a few
hours. The first algorithm is implemented via the ‘aln’ command, which
finds the suffix array (SA) coordinates of good hits of each individual
read, and the ‘samse/sampe’ command, which converts SA coordinates to
chromosomal coordinate and pairs reads (for ‘sampe’). The second
algorithm is invoked by the ‘dbtwsw’ command. It works for single-end
reads only.
COMMANDS AND OPTIONS
index bwa index [-p prefix] [-a algoType] [-c] <in.db.fasta>
Index database sequences in the FASTA format.
OPTIONS:
-c Build color-space index. The input fast should be in
nucleotide space.
-p STR Prefix of the output database [same as db filename]
-a STR Algorithm for constructing BWT index. Available
options are:
is IS linear-time algorithm for constructing
suffix array. It requires 5.37N memory where N
is the size of the database. IS is moderately
fast, but does not work with database larger
than 2GB. IS is the default algorithm due to
its simplicity. The current codes for IS
algorithm are reimplemented by Yuta Mori.
bwtsw Algorithm implemented in BWT-SW. This method
works with the whole human genome, but it does
not work with database smaller than 10MB and it
is usually slower than IS.
aln bwa aln [-n maxDiff] [-o maxGapO] [-e maxGapE] [-d nDelTail] [-i
nIndelEnd] [-k maxSeedDiff] [-l seedLen] [-t nThrds] [-cRN] [-M
misMsc] [-O gapOsc] [-E gapEsc] [-q trimQual] <in.db.fasta>
<in.query.fq> > <out.sai>
Find the SA coordinates of the input reads. Maximum maxSeedDiff
differences are allowed in the first seedLen subsequence and
maximum maxDiff differences are allowed in the whole sequence.
OPTIONS:
-n NUM Maximum edit distance if the value is INT, or the
fraction of missing alignments given 2% uniform base
error rate if FLOAT. In the latter case, the maximum
edit distance is automatically chosen for different
read lengths. [0.04]
-o INT Maximum number of gap opens [1]
-e INT Maximum number of gap extensions, -1 for k-difference
mode (disallowing long gaps) [-1]
-d INT Disallow a long deletion within INT bp towards the
3’-end [16]
-i INT Disallow an indel within INT bp towards the ends [5]
-l INT Take the first INT subsequence as seed. If INT is
larger than the query sequence, seeding will be
disabled. For long reads, this option is typically
ranged from 25 to 35 for ‘-k 2’. [inf]
-k INT Maximum edit distance in the seed [2]
-t INT Number of threads (multi-threading mode) [1]
-M INT Mismatch penalty. BWA will not search for suboptimal
hits with a score lower than (bestScore-misMsc). [3]
-O INT Gap open penalty [11]
-E INT Gap extension penalty [4]
-R INT Proceed with suboptimal alignments if there are no
more than INT equally best hits. This option only
affects paired-end mapping. Increasing this threshold
helps to improve the pairing accuracy at the cost of
speed, especially for short reads (~32bp).
-c Reverse query but not complement it, which is required
for alignment in the color space.
-N Disable iterative search. All hits with no more than
maxDiff differences will be found. This mode is much
slower than the default.
-q INT Parameter for read trimming. BWA trims a read down to
argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l
is the original read length. [0]
samse bwa samse [-n maxOcc] <in.db.fasta> <in.sai> <in.fq> > <out.sam>
Generate alignments in the SAM format given single-end reads.
Repetitive hits will be randomly chosen.
OPTIONS:
-n INT Maximum number of alignments to output in the XA tag
for reads paired properly. If a read has more than INT
hits, the XA tag will not be written. [3]
sampe bwa sampe [-a maxInsSize] [-o maxOcc] [-n maxHitPaired] [-N
maxHitDis] [-P] <in.db.fasta> <in1.sai> <in2.sai> <in1.fq>
<in2.fq> > <out.sam>
Generate alignments in the SAM format given paired-end reads.
Repetitive read pairs will be placed randomly.
OPTIONS:
-a INT Maximum insert size for a read pair to be considered
being mapped properly. Since 0.4.5, this option is only
used when there are not enough good alignment to infer
the distribution of insert sizes. [500]
-o INT Maximum occurrences of a read for pairing. A read with
more occurrneces will be treated as a single-end read.
Reducing this parameter helps faster pairing. [100000]
-P Load the entire FM-index into memory to reduce disk
operations (base-space reads only). With this option, at
least 1.25N bytes of memory are required, where N is the
length of the genome.
-n INT Maximum number of alignments to output in the XA tag for
reads paired properly. If a read has more than INT hits,
the XA tag will not be written. [3]
-N INT Maximum number of alignments to output in the XA tag for
disconcordant read pairs (excluding singletons). If a
read has more than INT hits, the XA tag will not be
written. [10]
bwasw bwa bwasw [-a matchScore] [-b mmPen] [-q gapOpenPen] [-r
gapExtPen] [-t nThreads] [-w bandWidth] [-T thres] [-s hspIntv]
[-z zBest] [-N nHspRev] [-c thresCoef] <in.db.fasta> <in.fq>
Align query sequences in the <in.fq> file.
OPTIONS:
-a INT Score of a match [1]
-b INT Mismatch penalty [3]
-q INT Gap open penalty [5]
-r INT Gap extension penalty. The penalty for a contiguous
gap of size k is q+k*r. [2]
-t INT Number of threads in the multi-threading mode [1]
-w INT Band width in the banded alignment [33]
-T INT Minimum score threshold divided by a [37]
-c FLOAT Coefficient for threshold adjustment according to
query length. Given an l-long query, the threshold for
a hit to be retained is a*max{T,c*log(l)}. [5.5]
-z INT Z-best heuristics. Higher -z increases accuracy at the
cost of speed. [1]
-s INT Maximum SA interval size for initiating a seed. Higher
-s increases accuracy at the cost of speed. [3]
-N INT Minimum number of seeds supporting the resultant
alignment to skip reverse alignment. [5]
SAM ALIGNMENT FORMAT
The output of the ‘aln’ command is binary and designed for BWA use
only. BWA outputs the final alignment in the SAM (Sequence
Alignment/Map) format. Each line consists of:
+----+-------+----------------------------------------------------------+
|Col | Field | Description |
+----+-------+----------------------------------------------------------+
| 1 | QNAME | Query (pair) NAME |
| 2 | FLAG | bitwise FLAG |
| 3 | RNAME | Reference sequence NAME |
| 4 | POS | 1-based leftmost POSition/coordinate of clipped sequence |
| 5 | MAPQ | MAPping Quality (Phred-scaled) |
| 6 | CIAGR | extended CIGAR string |
| 7 | MRNM | Mate Reference sequence NaMe (‘=’ if same as RNAME) |
| 8 | MPOS | 1-based Mate POSistion |
| 9 | ISIZE | Inferred insert SIZE |
|10 | SEQ | query SEQuence on the same strand as the reference |
|11 | QUAL | query QUALity (ASCII-33 gives the Phred base quality) |
|12 | OPT | variable OPTional fields in the format TAG:VTYPE:VALUE |
+----+-------+----------------------------------------------------------+
Each bit in the FLAG field is defined as:
+----+--------+---------------------------------------+
|Chr | Flag | Description |
+----+--------+---------------------------------------+
| p | 0x0001 | the read is paired in sequencing |
| P | 0x0002 | the read is mapped in a proper pair |
| u | 0x0004 | the query sequence itself is unmapped |
| U | 0x0008 | the mate is unmapped |
| r | 0x0010 | strand of the query (1 for reverse) |
| R | 0x0020 | strand of the mate |
| 1 | 0x0040 | the read is the first read in a pair |
| 2 | 0x0080 | the read is the second read in a pair |
| s | 0x0100 | the alignment is not primary |
| f | 0x0200 | QC failure |
| d | 0x0400 | optical or PCR duplicate |
+----+--------+---------------------------------------+
The Please check <http://samtools.sourceforge.net> for the format
specification and the tools for post-processing the alignment.
BWA generates the following optional fields. Tags starting with ‘X’ are
specific to BWA.
+----+------------------------------------------------+
|Tag | Meaning |
+----+------------------------------------------------+
|NM | Edit distance |
|MD | Mismatching positions/bases |
|AS | Alignment score |
+----+------------------------------------------------+
|X0 | Number of best hits |
|X1 | Number of suboptimal hits found by BWA |
|XN | Number of ambiguous bases in the referenece |
|XM | Number of mismatches in the alignment |
|XO | Number of gap opens |
|XG | Number of gap extentions |
|XT | Type: Unique/Repeat/N/Mate-sw |
|XA | Alternative hits; format: (chr,pos,CIGAR,NM;)* |
+----+------------------------------------------------+
|XS | Suboptimal alignment score |
|XF | Support from forward/reverse alignment |
|XE | Number of supporting seeds |
+----+------------------------------------------------+
Note that XO and XG are generated by BWT search while the CIGAR string
by Smith-Waterman alignment. These two tags may be inconsistent with
the CIGAR string. This is not a bug.
NOTES ON SHORT-READ ALIGNMENT
Alignment Accuracy
When seeding is disabled, BWA guarantees to find an alignment
containing maximum maxDiff differences including maxGapO gap opens
which do not occur within nIndelEnd bp towards either end of the query.
Longer gaps may be found if maxGapE is positive, but it is not
guaranteed to find all hits. When seeding is enabled, BWA further
requires that the first seedLen subsequence contains no more than
maxSeedDiff differences.
When gapped alignment is disabled, BWA is expected to generate the same
alignment as Eland, the Illumina alignment program. However, as BWA
change ‘N’ in the database sequence to random nucleotides, hits to
these random sequences will also be counted. As a consequence, BWA may
mark a unique hit as a repeat, if the random sequences happen to be
identical to the sequences which should be unqiue in the database. This
random behaviour will be avoided in future releases.
By default, if the best hit is no so repetitive (controlled by -R), BWA
also finds all hits contains one more mismatch; otherwise, BWA finds
all equally best hits only. Base quality is NOT considered in
evaluating hits. In paired-end alignment, BWA pairs all hits it found.
It further performs Smith-Waterman alignment for unmapped reads with
mates mapped to rescue mapped mates, and for high-quality anomalous
pairs to fix potential alignment errors.
Estimating Insert Size Distribution
BWA estimates the insert size distribution per 256*1024 read pairs. It
first collects pairs of reads with both ends mapped with a single-end
quality 20 or higher and then calculates median (Q2), lower and higher
quartile (Q1 and Q3). It estimates the mean and the variance of the
insert size distribution from pairs whose insert sizes are within
interval [Q1-2(Q3-Q1), Q3+2(Q3-Q1)]. The maximum distance x for a pair
considered to be properly paired (SAM flag 0x2) is calculated by
solving equation Phi((x-mu)/sigma)=x/L*p0, where mu is the mean, sigma
is the standard error of the insert size distribution, L is the length
of the genome, p0 is prior of anomalous pair and Phi() is the standard
cumulative distribution function. For mapping Illumina short-insert
reads to the human genome, x is about 6-7 sigma away from the mean.
Quartiles, mean, variance and x will be printed to the standard error
output.
Memory Requirement
With bwtsw algorithm, 2.5GB memory is required for indexing the
complete human genome sequences. For short reads, the ‘aln’ command
uses ~2.3GB memory and the ‘sampe’ command uses ~3.5GB.
Speed
Indexing the human genome sequences takes 3 hours with bwtsw algorithm.
Indexing smaller genomes with IS or divsufsort algorithms is several
times faster, but requires more memory.
Speed of alignment is largely determined by the error rate of the query
sequences (r). Firstly, BWA runs much faster for near perfect hits than
for hits with many differences, and it stops searching for a hit with
l+2 differences if a l-difference hit is found. This means BWA will be
very slow if r is high because in this case BWA has to visit hits with
many differences and looking for these hits is expensive. Secondly, the
alignment algorithm behind makes the speed sensitive to [k log(N)/m],
where k is the maximum allowed differences, N the size of database and
m the length of a query. In practice, we choose k w.r.t. r and
therefore r is the leading factor. I would not recommend to use BWA on
data with r>0.02.
Pairing is slower for shorter reads. This is mainly because shorter
reads have more spurious hits and converting SA coordinates to
chromosomal coordinates are very costly.
In a practical experiment, BWA is able to map 2 million 32bp reads to a
bacterial genome in several minutes, map the same amount of reads to
human X chromosome in 8-15 minutes and to the human genome in 15-25
minutes. This result implies that the speed of BWA is insensitive to
the size of database and therefore BWA is more efficient when the
database is sufficiently large. On smaller genomes, hash based
algorithms are usually much faster.
NOTES ON LONG-READ ALIGNMENT
Command ‘bwasw’ is designed for long-read alignment. The algorithm
behind, BWA-SW, is similar to BWT-SW, but does not guarantee to find
all local hits due to the heuristic acceleration. It tends to be faster
and more accurate if the resultant alignment is supported by more
seeds, and therefore BWA-SW usually performs better on long queries
than on short ones.
On 350-1000bp reads, BWA-SW is several to tens of times faster than the
existing programs. Its accuracy is comparable to SSAHA2, more accurate
than BLAT. Like BLAT, BWA-SW also finds chimera which may pose a
challenge to SSAHA2. On 10-100kbp queries where chimera detection is
important, BWA-SW is over 10X faster than BLAT while being more
sensitive.
BWA-SW can also be used to align ~100bp reads, but it is slower than
the short-read algorithm. Its sensitivity and accuracy is lower than
SSAHA2 especially when the sequencing error rate is above 2%. This is
the trade-off of the 30X speed up in comparison to SSAHA2’s -454 mode.
SEE ALSO
BWA website <http://bio-bwa.sourceforge.net>, Samtools website
<http://samtools.sourceforge.net>
AUTHOR
Heng Li at the Sanger Institute wrote the key source codes and
integrated the following codes for BWT construction: bwtsw
<http://i.cs.hku.hk/~ckwong3/bwtsw/>, implemented by Chi-Kwong Wong at
the University of Hong Kong and IS
<http://yuta.256.googlepages.com/sais> originally proposed by Nong Ge
<http://www.cs.sysu.edu.cn/nong/> at the Sun Yat-Sen University and
implemented by Yuta Mori.
LICENSE AND CITATION
The full BWA package is distributed under GPLv3 as it uses source codes
from BWT-SW which is covered by GPL. Sorting, hash table, BWT and IS
libraries are distributed under the MIT license.
If you use the short-read alignment component, please cite the
following paper:
Li H. and Durbin R. (2009) Fast and accurate short read alignment with
Burrows-Wheeler transform. Bioinformatics, 25, 1754-60. [PMID:
19451168]
If you use the long-read component (BWA-SW), please cite:
Li H. and Durbin R. (2010) Fast and accurate long-read alignment with
Burrows-Wheeler transform. Bioinformatics. [PMID: 20080505]
HISTORY
BWA is largely influenced by BWT-SW. It uses source codes from BWT-SW
and mimics its binary file formats; BWA-SW resembles BWT-SW in several
ways. The initial idea about BWT-based alignment also came from the
group who developed BWT-SW. At the same time, BWA is different enough
from BWT-SW. The short-read alignment algorithm bears no similarity to
Smith-Waterman algorithm any more. While BWA-SW learns from BWT-SW, it
introduces heuristics that can hardly be applied to the original
algorithm. In all, BWA does not guarantee to find all local hits as
what BWT-SW is designed to do, but it is much faster than BWT-SW on
both short and long query sequences.
I started to write the first piece of codes on 24 May 2008 and got the
initial stable version on 02 June 2008. During this period, I was
acquainted that Professor Tak-Wah Lam, the first author of BWT-SW
paper, was collaborating with Beijing Genomics Institute on SOAP2, the
successor to SOAP (Short Oligonucleotide Analysis Package). SOAP2 has
come out in November 2008. According to the SourceForge download page,
the third BWT-based short read aligner, bowtie, was first released in
August 2008. At the time of writing this manual, at least three more
BWT-based short-read aligners are being implemented.
The BWA-SW algorithm is a new component of BWA. It was conceived in
November 2008 and implemented ten months later.