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bwa - Burrows-Wheeler Alignment Tool


bwa index ref.fa

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

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

bwa aln ref.fa short_read.fq > aln_sa.sai

bwa samse ref.fa aln_sa.sai short_read.fq > aln-se.sam

bwa sampe ref.fa aln_sa1.sai aln_sa2.sai read1.fq read2.fq > aln-pe.sam

bwa bwasw ref.fa long_read.fq > aln.sam


BWA is a software package for mapping low-divergent 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 1Mbp. BWA-MEM and BWA-SW share
similar features such as long-read support and split alignment, but BWA-MEM, which is the
latest, is generally recommended for high-quality queries as it is faster and more
accurate. BWA-MEM also has better performance than BWA-backtrack for 70-100bp Illumina

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.


index bwa index [-p prefix] [-a algoType] db.fa

Index database sequences in the FASTA format.


-p STR Prefix of the output database [same as db filename]

-a STR Algorithm for constructing BWT index. BWA implements two algorithms for
BWT construction: is and bwtsw. The first algorithm is a little faster
for small database but requires large RAM and does not work for databases
with total length longer than 2GB. The second algorithm is adapted from
the BWT-SW source code. It in theory works with database with trillions
of bases. When this option is not specified, the appropriate algorithm
will be chosen automatically.

mem bwa mem [-aCHjMpP] [-t nThreads] [-k minSeedLen] [-w bandWidth] [-d zDropoff] [-r
seedSplitRatio] [-c maxOcc] [-D chainShadow] [-m maxMateSW] [-W minSeedMatch] [-A
matchScore] [-B mmPenalty] [-O gapOpenPen] [-E gapExtPen] [-L clipPen] [-U
unpairPen] [-R RGline] [-H HDlines] [-v verboseLevel] db.prefix reads.fq [mates.fq]

Align 70bp-1Mbp query sequences with the BWA-MEM algorithm. Briefly, the algorithm
works by seeding alignments with maximal exact matches (MEMs) and then extending
seeds with the affine-gap Smith-Waterman algorithm (SW).

If mates.fq file is absent and option -p is not set, this command regards input
reads are single-end. If mates.fq is present, this command assumes the i-th read in
reads.fq and the i-th read in mates.fq constitute a read pair. If -p is used, the
command assumes the 2i-th and the (2i+1)-th read in reads.fq constitute a read pair
(such input file is said to be interleaved). In this case, mates.fq is ignored. In
the paired-end mode, the mem command will infer the read orientation and the insert
size distribution from a batch of reads.

The BWA-MEM algorithm performs local alignment. It may produce multiple primary
alignments for different part of a query sequence. This is a crucial feature for
long sequences. However, some tools such as Picard's markDuplicates does not work
with split alignments. One may consider to use option -M to flag shorter split hits
as secondary.


-t INT Number of threads [1]

-k INT Minimum seed length. Matches shorter than INT will be missed. The
alignment speed is usually insensitive to this value unless it
significantly deviates from 20. [19]

-w INT Band width. Essentially, gaps longer than INT will not be found. Note
that the maximum gap length is also affected by the scoring matrix and
the hit length, not solely determined by this option. [100]

-d INT Off-diagonal X-dropoff (Z-dropoff). Stop extension when the difference
between the best and the current extension score is above |i-j|*A+INT,
where i and j are the current positions of the query and reference,
respectively, and A is the matching score. Z-dropoff is similar to
BLAST's X-dropoff except that it doesn't penalize gaps in one of the
sequences in the alignment. Z-dropoff not only avoids unnecessary
extension, but also reduces poor alignments inside a long good alignment.

-r FLOAT Trigger re-seeding for a MEM longer than minSeedLen*FLOAT. This is a key
heuristic parameter for tuning the performance. Larger value yields fewer
seeds, which leads to faster alignment speed but lower accuracy. [1.5]

-c INT Discard a MEM if it has more than INT occurence in the genome. This is an
insensitive parameter. [500]

-D INT Drop chains shorter than FLOAT fraction of the longest overlapping chain

-m INT Perform at most INT rounds of mate-SW [50]

-W INT Drop a chain if the number of bases in seeds is smaller than INT. This
option is primarily used for longer contigs/reads. When positive, it also
affects seed filtering. [0]

-P In the paired-end mode, perform SW to rescue missing hits only but do not
try to find hits that fit a proper pair.


-A INT Matching score. [1]

-B INT Mismatch penalty. The sequence error rate is approximately: {.75 *
exp[-log(4) * B/A]}. [4]

Gap open penalty. If two numbers are specified, the first is the penalty
of openning a deletion and the second for openning an insertion. [6]

Gap extension penalty. If two numbers are specified, the first is the
penalty of extending a deletion and second for extending an insertion. A
gap of length k costs O + k*E (i.e. -O is for opening a zero-length
gap). [1]

Clipping penalty. When performing SW extension, BWA-MEM keeps track of
the best score reaching the end of query. If this score is larger than
the best SW score minus the clipping penalty, clipping will not be
applied. Note that in this case, the SAM AS tag reports the best SW
score; clipping penalty is not deduced. If two numbers are provided, the
first is for 5'-end clipping and second for 3'-end clipping. [5]

-U INT Penalty for an unpaired read pair. BWA-MEM scores an unpaired read pair
as scoreRead1+scoreRead2-INT and scores a paired as
scoreRead1+scoreRead2-insertPenalty. It compares these two scores to
determine whether we should force pairing. A larger value leads to more
aggressive read pair. [17]


-p Smart pairing. If two adjacent reads have the same name, they are
considered to form a read pair. This way, paired-end and single-end reads
can be mixed in a single FASTA/Q stream.

-R STR Complete read group header line. '\t' can be used in STR and will be
converted to a TAB in the output SAM. The read group ID will be attached
to every read in the output. An example is '@RG\tID:foo\tSM:bar'. [null]

-H ARG If ARG starts with @, it is interpreted as a string and gets inserted
into the output SAM header; otherwise, ARG is interpreted as a file with
all lines starting with @ in the file inserted into the SAM header.

-T INT Don't output alignment with score lower than INT. This option affects
output and occasionally SAM flag 2. [30]

-j Treat ALT contigs as part of the primary assembly (i.e. ignore the
db.prefix.alt file).

-h INT[,INT2]
If a query has not more than INT hits with score higher than 80% of the
best hit, output them all in the XA tag. If INT2 is specified, BWA-MEM
outputs up to INT2 hits if the list contains a hit to an ALT contig.

-a Output all found alignments for single-end or unpaired paired-end reads.
These alignments will be flagged as secondary alignments.

-C Append append FASTA/Q comment to SAM output. This option can be used to
transfer read meta information (e.g. barcode) to the SAM output. Note
that the FASTA/Q comment (the string after a space in the header line)
must conform the SAM spec (e.g. BC:Z:CGTAC). Malformated comments lead to
incorrect SAM output.

-Y Use soft clipping CIGAR operation for supplementary alignments. By
default, BWA-MEM uses soft clipping for the primary alignment and hard
clipping for supplementary alignments.

-M Mark shorter split hits as secondary (for Picard compatibility).

-v INT Control the verbose level of the output. This option has not been fully
supported throughout BWA. Ideally, a value 0 for disabling all the output
to stderr; 1 for outputting errors only; 2 for warnings and errors; 3 for
all normal messages; 4 or higher for debugging. When this option takes
value 4, the output is not SAM. [3]

Specify the mean, standard deviation (10% of the mean if absent), max (4
sigma from the mean if absent) and min (4 sigma if absent) of the insert
size distribution. Only applicable to the FR orientation. By default,
BWA-MEM infers these numbers and the pair orientations given enough
reads. [inferred]

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.


-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. (Disabled since 0.6.x)

-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]

-I The input is in the Illumina 1.3+ read format (quality equals ASCII-64).

-B INT Length of barcode starting from the 5'-end. When INT is positive, the
barcode of each read will be trimmed before mapping and will be written
at the BC SAM tag. For paired-end reads, the barcode from both ends are
concatenated. [0]

-b Specify the input read sequence file is the BAM format. For paired-end
data, two ends in a pair must be grouped together and options -1 or -2
are usually applied to specify which end should be mapped. Typical
command lines for mapping pair-end data in the BAM format are:

bwa aln ref.fa -b1 reads.bam > 1.sai
bwa aln ref.fa -b2 reads.bam > 2.sai
bwa sampe ref.fa 1.sai 2.sai reads.bam reads.bam > aln.sam

-0 When -b is specified, only use single-end reads in mapping.

-1 When -b is specified, only use the first read in a read pair in mapping
(skip single-end reads and the second reads).

-2 When -b is specified, only use the second read in a read pair in mapping.

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.


-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]

-r STR Specify the read group in a format like `@RG\tID:foo\tSM:bar'. [null]

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.


-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.

-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]

-r STR Specify the read group in a format like `@RG\tID:foo\tSM:bar'. [null]

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> [mate.fq]

Align query sequences in the in.fq file. When mate.fq is present, perform paired-
end alignment. The paired-end mode only works for reads Illumina short-insert
libraries. In the paired-end mode, BWA-SW may still output split alignments but
they are all marked as not properly paired; the mate positions will not be written
if the mate has multiple local hits.


-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]


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:

│ 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:

│ 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 │
│ S │ 0x0800 │ supplementary alignment │

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.

NM │ Edit distance │
MD │ Mismatching positions/bases │
AS │ Alignment score │
BC │ Barcode sequence │
SA │ Supplementary alignments │
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.


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
version 1, 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.

By default, if the best hit is not highly 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 the paired-end mode, BWA pairs all hits
it found. It further performs Smith-Waterman alignment for unmapped reads to rescue reads
with a high erro rate, and for high-quality anomalous pairs to fix potential alignment

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, 5GB memory is required for indexing the complete human genome
sequences. For short reads, the aln command uses ~3.2GB memory and the sampe command uses

Indexing the human genome sequences takes 3 hours with bwtsw algorithm. Indexing smaller
genomes with IS algorithms is faster, but requires more memory.

The 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.


Since version 0.6, BWA has been able to work with a reference genome longer than 4GB.
This feature makes it possible to integrate the forward and reverse complemented genome in
one FM-index, which speeds up both BWA-short and BWA-SW. As a tradeoff, BWA uses more
memory because it has to keep all positions and ranks in 64-bit integers, twice larger
than 32-bit integers used in the previous versions.

The latest BWA-SW also works for paired-end reads longer than 100bp. In comparison to BWA-
short, BWA-SW tends to be more accurate for highly unique reads and more robust to
relative long INDELs and structural variants. Nonetheless, BWA-short usually has higher
power to distinguish the optimal hit from many suboptimal hits. The choice of the mapping
algorithm may depend on the application.

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