BWA操作手册

Manual Reference Pages - bwa

1.

NAME

bwa - Burrows-Wheeler Alignment Tool

2.

CONTENTS

(1) Synopsis

(2) Description

(3) Commands And Options

(4) Sam Alignment Format

(5) Notes On Short-read Alignment

Alignment Accuracy

Estimating Insert Size Distribution

Memory Requirement

Speed

(6) Changes In Bwa-0.6

(7) License And Citation

(1)

SYNOPSIS

bwa index

bwa mem >

bwa mem >

bwa aln short_ > aln_

bwa samse aln_ short_ >

bwa sampe aln_ aln_ >

bwa bwasw long_ >

(2)

DESCRIPTION

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

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.

(3)

COMMANDS AND OPTIONS

index

bwa index [-p prefix] [-a algoType] <>

Index database sequences in the FASTA format.

OPTIONS:

-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.

mem

bwa mem

[

-aCHMpP

] [-

t

nThreads] [-

k

minSeedLen] [-

w

bandWidth] [-

d

zDropoff] [-

r

seedSplitRatio] [-

c

maxOcc] [-

A

matchScore] [-

B

mmPenalty]

[-

O

gapOpenPen] [-

E

gapExtPen] [-

L

clipPen] [-

U

unpairPen] [-

R

RGline]

[-

v

verboseLevel] []

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 file is absent and option -

p

is not set, this command regards input

reads

are single-end.

If

is

present,

this

command assumes the i-th

read in and the i-th read in constitute a read pair. If

-

p

is

used,

the

command

assumes

the

2i-th

and

the

(2i+1)-th

read

in

constitute a read pair (such input file is said to be interleaved). In this case,

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.

OPTIONS:

-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 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. [100]

-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. [10000]

-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]

-O

INT

Gap open penalty. [6]

-E

INT

Gap extension penalty. A gap of length k costs O + k*E (i.e. -O is for

opening a zero-length gap). [1]

-L

INT

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 deducted. [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. [9]

-p

Assume the first input query file is interleaved paired-end FASTA/Q.

See the command description for details.

-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]

-T

INT

Don’t output alignment with score lower than INT. This option only

affects output. [30]

-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.

-H

Use

hard

clipping

’H’

in

the

SAM

output.

This

option

may

dramatically

reduce

the

redundancy

of

output

when

mapping

long

contig or BAC sequences.

-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]

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]

<>

<>

>

<>

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

M

aximum 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

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 -b1 >

bwa aln -b2 >

bwa sampe >

-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] <> <> <> > <>

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]

-r

STR

Specify

the

read

group

in

a

format

like

‘@RG< /p>

tID:foo

tSM:bar’.

[null]

sampe

bwa sampe [-a maxInsSize] [-o maxOcc] [-n maxHitPaired] [-N maxHitDis]

[-P] <> <> <> <> <> > <>

Generate alignments in

the SAM format given paired-end reads. Repetitive

read pairs will be placed randomly.