samtools.1

.TH samtools 1 "2 December 2010" "samtools-0.1.12" "Bioinformatics tools"
.SH NAME
.PP
samtools - Utilities for the Sequence Alignment/Map (SAM) format
.SH SYNOPSIS
.PP
samtools view -bt ref_list.txt -o aln.bam aln.sam.gz
.PP
samtools sort aln.bam aln.sorted
.PP
samtools index aln.sorted.bam
.PP
samtools idxstats aln.sorted.bam
.PP
samtools view aln.sorted.bam chr2:20,100,000-20,200,000
.PP
samtools merge out.bam in1.bam in2.bam in3.bam
.PP
samtools faidx ref.fasta
.PP
samtools pileup -vcf ref.fasta aln.sorted.bam
.PP
samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
.PP
samtools tview aln.sorted.bam ref.fasta

.SH DESCRIPTION
.PP
Samtools is a set of utilities that manipulate alignments in the BAM
format. It imports from and exports to the SAM (Sequence Alignment/Map)
format, does sorting, merging and indexing, and allows to retrieve reads
in any regions swiftly.

Samtools is designed to work on a stream. It regards an input file `-'
as the standard input (stdin) and an output file `-' as the standard
output (stdout). Several commands can thus be combined with Unix
pipes. Samtools always output warning and error messages to the standard
error output (stderr).

Samtools is also able to open a BAM (not SAM) file on a remote FTP or
HTTP server if the BAM file name starts with `ftp://' or `http://'.
Samtools checks the current working directory for the index file and
will download the index upon absence. Samtools does not retrieve the
entire alignment file unless it is asked to do so.

.SH COMMANDS AND OPTIONS

.TP 10
.B view
samtools view [-bchuHS] [-t in.refList] [-o output] [-f reqFlag] [-F
skipFlag] [-q minMapQ] [-l library] [-r readGroup] [-R rgFile] <in.bam>|<in.sam> [region1 [...]]

Extract/print all or sub alignments in SAM or BAM format. If no region
is specified, all the alignments will be printed; otherwise only
alignments overlapping the specified regions will be output. An
alignment may be given multiple times if it is overlapping several
regions. A region can be presented, for example, in the following
format: `chr2' (the whole chr2), `chr2:1000000' (region starting from
1,000,000bp) or `chr2:1,000,000-2,000,000' (region between 1,000,000 and
2,000,000bp including the end points). The coordinate is 1-based.

.B OPTIONS:
.RS
.TP 8
.B -b
Output in the BAM format.
.TP
.BI -f \ INT
Only output alignments with all bits in INT present in the FLAG
field. INT can be in hex in the format of /^0x[0-9A-F]+/ [0]
.TP
.BI -F \ INT
Skip alignments with bits present in INT [0]
.TP
.B -h
Include the header in the output.
.TP
.B -H
Output the header only.
.TP
.BI -l \ STR
Only output reads in library STR [null]
.TP
.BI -o \ FILE
Output file [stdout]
.TP
.BI -q \ INT
Skip alignments with MAPQ smaller than INT [0]
.TP
.BI -r \ STR
Only output reads in read group STR [null]
.TP
.BI -R \ FILE
Output reads in read groups listed in
.I FILE
[null]
.TP
.B -S
Input is in SAM. If @SQ header lines are absent, the
.B `-t'
option is required.
.TP
.B -c
Instead of printing the alignments, only count them and print the
total number. All filter options, such as
.B `-f',
.B `-F'
and
.B `-q'
, are taken into account.
.TP
.BI -t \ FILE
This file is TAB-delimited. Each line must contain the reference name
and the length of the reference, one line for each distinct reference;
additional fields are ignored. This file also defines the order of the
reference sequences in sorting. If you run `samtools faidx <ref.fa>',
the resultant index file
.I <ref.fa>.fai
can be used as this
.I <in.ref_list>
file.
.TP
.B -u
Output uncompressed BAM. This option saves time spent on
compression/decomprssion and is thus preferred when the output is piped
to another samtools command.
.RE

.TP
.B tview
samtools tview <in.sorted.bam> [ref.fasta]

Text alignment viewer (based on the ncurses library). In the viewer,
press `?' for help and press `g' to check the alignment start from a
region in the format like `chr10:10,000,000' or `=10,000,000' when
viewing the same reference sequence.

.TP
.B mpileup
samtools mpileup [-EBug] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-M capMapQ] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]

Generate BCF or pileup for one or multiple BAM files. Alignment records
are grouped by sample identifiers in @RG header lines. If sample
identifiers are absent, each input file is regarded as one sample.

.B OPTIONS:
.RS
.TP 8
.B -B
Disable probabilistic realignment for the computation of base alignment
quality (BAQ). BAQ is the Phred-scaled probability of a read base being
misaligned. Applying this option greatly helps to reduce false SNPs
caused by misalignments.
.TP
.BI -C \ INT
Coefficient for downgrading mapping quality for reads containing
excessive mismatches. Given a read with a phred-scaled probability q of
being generated from the mapped position, the new mapping quality is
about sqrt((INT-q)/INT)*INT. A zero value disables this
functionality; if enabled, the recommended value for BWA is 50. [0]
.TP
.BI -e \ INT
Phred-scaled gap extension sequencing error probability. Reducing
.I INT
leads to longer indels. [20]
.TP
.B -E
Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt
specificity a little bit.
.TP
.BI -f \ FILE
The reference file [null]
.TP
.B -g
Compute genotype likelihoods and output them in the binary call format (BCF).
.TP
.BI -h \ INT
Coefficient for modeling homopolymer errors. Given an
.IR l -long
homopolymer
run, the sequencing error of an indel of size
.I s
is modeled as
.IR INT * s / l .
[100]
.TP
.BI -l \ FILE
File containing a list of sites where pileup or BCF is outputted [null]
.TP
.BI -o \ INT
Phred-scaled gap open sequencing error probability. Reducing
.I INT
leads to more indel calls. [40]
.TP
.BI -P \ STR
Comma dilimited list of platforms (determined by
.BR @RG-PL )
from which indel candidates are obtained. It is recommended to collect
indel candidates from sequencing technologies that have low indel error
rate such as ILLUMINA. [all]
.TP
.BI -q \ INT
Minimum mapping quality for an alignment to be used [0]
.TP
.BI -Q \ INT
Minimum base quality for a base to be considered [13]
.TP
.BI -r \ STR
Only generate pileup in region
.I STR
[all sites]
.TP
.B -u
Similar to
.B -g
except that the output is uncompressed BCF, which is preferred for piping.
.RE

.TP
.B reheader
samtools reheader <in.header.sam> <in.bam>

Replace the header in
.I in.bam
with the header in
.I in.header.sam.
This command is much faster than replacing the header with a
BAM->SAM->BAM conversion.

.TP
.B sort
samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>

Sort alignments by leftmost coordinates. File
.I <out.prefix>.bam
will be created. This command may also create temporary files
.I <out.prefix>.%d.bam
when the whole alignment cannot be fitted into memory (controlled by
option -m).

.B OPTIONS:
.RS
.TP 8
.B -o
Output the final alignment to the standard output.
.TP
.B -n
Sort by read names rather than by chromosomal coordinates
.TP
.BI -m \ INT
Approximately the maximum required memory. [500000000]
.RE

.TP
.B merge
samtools merge [-nur] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]

Merge multiple sorted alignments.
The header reference lists of all the input BAM files, and the @SQ headers of
.IR inh.sam ,
if any, must all refer to the same set of reference sequences.
The header reference list and (unless overridden by
.BR -h )
`@' headers of
.I in1.bam
will be copied to
.IR out.bam ,
and the headers of other files will be ignored.

.B OPTIONS:
.RS
.TP 8
.BI -h \ FILE
Use the lines of
.I FILE
as `@' headers to be copied to
.IR out.bam ,
replacing any header lines that would otherwise be copied from
.IR in1.bam .
.RI ( FILE
is actually in SAM format, though any alignment records it may contain
are ignored.)
.TP
.BI -R \ STR
Merge files in the specified region indicated by
.I STR
.TP
.B -r
Attach an RG tag to each alignment. The tag value is inferred from file names.
.TP
.B -n
The input alignments are sorted by read names rather than by chromosomal
coordinates
.TP
.B -u
Uncompressed BAM output
.RE

.TP
.B index
samtools index <aln.bam>

Index sorted alignment for fast random access. Index file
.I <aln.bam>.bai
will be created.

.TP
.B idxstats
samtools idxstats <aln.bam>

Retrieve and print stats in the index file. The output is TAB delimited
with each line consisting of reference sequence name, sequence length, #
mapped reads and # unmapped reads.

.TP
.B faidx
samtools faidx <ref.fasta> [region1 [...]]

Index reference sequence in the FASTA format or extract subsequence from
indexed reference sequence. If no region is specified,
.B faidx
will index the file and create
.I <ref.fasta>.fai
on the disk. If regions are speficified, the subsequences will be
retrieved and printed to stdout in the FASTA format. The input file can
be compressed in the
.B RAZF
format.

.TP
.B fixmate
samtools fixmate <in.nameSrt.bam> <out.bam>

Fill in mate coordinates, ISIZE and mate related flags from a
name-sorted alignment.

.TP
.B rmdup
samtools rmdup [-sS] <input.srt.bam> <out.bam>

Remove potential PCR duplicates: if multiple read pairs have identical
external coordinates, only retain the pair with highest mapping quality.
In the paired-end mode, this command
.B ONLY
works with FR orientation and requires ISIZE is correctly set. It does
not work for unpaired reads (e.g. two ends mapped to different
chromosomes or orphan reads).

.B OPTIONS:
.RS
.TP 8
.B -s
Remove duplicate for single-end reads. By default, the command works for
paired-end reads only.
.TP 8
.B -S
Treat paired-end reads and single-end reads.
.RE

.TP
.B calmd
samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta>

Generate the MD tag. If the MD tag is already present, this command will
give a warning if the MD tag generated is different from the existing
tag. Output SAM by default.

.B OPTIONS:
.RS
.TP 8
.B -A
When used jointly with
.B -r
this option overwrites the original base quality.
.TP 8
.B -e
Convert a the read base to = if it is identical to the aligned reference
base. Indel caller does not support the = bases at the moment.
.TP
.B -u
Output uncompressed BAM
.TP
.B -b
Output compressed BAM
.TP
.B -S
The input is SAM with header lines
.TP
.BI -C \ INT
Coefficient to cap mapping quality of poorly mapped reads. See the
.B pileup
command for details. [0]
.TP
.B -r
Compute the BQ tag (without -A) or cap base quality by BAQ (with -A).
.TP
.B -E
Extended BAQ calculation. This option trades specificity for sensitivity, though the
effect is minor.
.RE

.TP
.B targetcut
samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam>

This command identifies target regions by examining the continuity of read depth, computes
haploid consensus sequences of targets and outputs a SAM with each sequence corresponding
to a target. When option
.B -f
is in use, BAQ will be applied. This command is
.B only
designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].
.RE

.TP
.B phase
samtools phase [-F] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam>

Call and phase heterozygous SNPs.
.B OPTIONS:
.RS
.TP 8
.BI -b \ STR
Prefix of BAM output. When this option is in use, phase-0 reads will be saved in file
.BR STR .0.bam
and phase-1 reads in
.BR STR .1.bam.
Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads
with switch errors will be saved in
.BR STR .chimeric.bam.
[null]
.TP
.B -F
Do not attempt to fix chimeric reads.
.TP
.BI -k \ INT
Maximum length for local phasing. [13]
.TP
.BI -q \ INT
Minimum Phred-scaled LOD to call a heterozygote. [40]
.TP
.BI -Q \ INT
Minimum base quality to be used in het calling. [13]
.RE

.TP
.B pileup
samtools pileup [-2sSBicv] [-f in.ref.fasta] [-t in.ref_list] [-l
in.site_list] [-C capMapQ] [-M maxMapQ] [-T theta] [-N nHap] [-r
pairDiffRate] [-m mask] [-d maxIndelDepth] [-G indelPrior]
<in.bam>|<in.sam>

Print the alignment in the pileup format. In the pileup format, each
line represents a genomic position, consisting of chromosome name,
coordinate, reference base, read bases, read qualities and alignment
mapping qualities. Information on match, mismatch, indel, strand,
mapping quality and start and end of a read are all encoded at the read
base column. At this column, a dot stands for a match to the reference
base on the forward strand, a comma for a match on the reverse strand,
a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward
strand and `acgtn' for a mismatch on the reverse strand. A pattern
`\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this
reference position and the next reference position. The length of the
insertion is given by the integer in the pattern, followed by the
inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+'
represents a deletion from the reference. The deleted bases will be
presented as `*' in the following lines. Also at the read base column, a
symbol `^' marks the start of a read. The ASCII of the character
following `^' minus 33 gives the mapping quality. A symbol `$' marks the
end of a read segment.

If option
.B -c
is applied, the consensus base, Phred-scaled consensus quality, SNP
quality (i.e. the Phred-scaled probability of the consensus being
identical to the reference) and root mean square (RMS) mapping quality
of the reads covering the site will be inserted between the `reference
base' and the `read bases' columns. An indel occupies an additional
line. Each indel line consists of chromosome name, coordinate, a star,
the genotype, consensus quality, SNP quality, RMS mapping quality, #
covering reads, the first alllele, the second allele, # reads supporting
the first allele, # reads supporting the second allele and # reads
containing indels different from the top two alleles.

.B NOTE:
Since 0.1.10, the `pileup' command is deprecated by `mpileup'.

.B OPTIONS:
.RS
.TP 10
.B -B
Disable the BAQ computation. See the
.B mpileup
command for details.
.TP
.B -c
Call the consensus sequence. Options
.BR -T ", " -N ", " -I " and " -r
are only effective when
.BR -c " or " -g
is in use.
.TP
.BI -C \ INT
Coefficient for downgrading the mapping quality of poorly mapped
reads. See the
.B mpileup
command for details. [0]
.TP
.BI -d \ INT
Use the first
.I NUM
reads in the pileup for indel calling for speed up. Zero for unlimited. [1024]
.TP
.BI -f \ FILE
The reference sequence in the FASTA format. Index file
.I FILE.fai
will be created if
absent.
.TP
.B -g
Generate genotype likelihood in the binary GLFv3 format. This option
suppresses -c, -i and -s. This option is deprecated by the
.B mpileup
command.
.TP
.B -i
Only output pileup lines containing indels.
.TP
.BI -I \ INT
Phred probability of an indel in sequencing/prep. [40]
.TP
.BI -l \ FILE
List of sites at which pileup is output. This file is space
delimited. The first two columns are required to be chromosome and
1-based coordinate. Additional columns are ignored. It is
recommended to use option
.TP
.BI -m \ INT
Filter reads with flag containing bits in
.I INT
[1796]
.TP
.BI -M \ INT
Cap mapping quality at INT [60]
.TP
.BI -N \ INT
Number of haplotypes in the sample (>=2) [2]
.TP
.BI -r \ FLOAT
Expected fraction of differences between a pair of haplotypes [0.001]
.TP
.B -s
Print the mapping quality as the last column. This option makes the
output easier to parse, although this format is not space efficient.
.TP
.B -S
The input file is in SAM.
.TP
.BI -t \ FILE
List of reference names ane sequence lengths, in the format described
for the
.B import
command. If this option is present, samtools assumes the input
.I <in.alignment>
is in SAM format; otherwise it assumes in BAM format.
.B -s
together with
.B -l
as in the default format we may not know the mapping quality.
.TP
.BI -T \ FLOAT
The theta parameter (error dependency coefficient) in the maq consensus
calling model [0.85]
.RE

.SH SAM FORMAT

SAM is TAB-delimited. Apart from the header lines, which are started
with the `@' symbol, each alignment line consists of:

.TS
center box;
cb | cb | cb
n | l | l .
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
.TE

.PP
Each bit in the FLAG field is defined as:

.TS
center box;
cb | cb | cb
l | c | l .
Flag	Chr	Description
_
0x0001	p	the read is paired in sequencing
0x0002	P	the read is mapped in a proper pair
0x0004	u	the query sequence itself is unmapped
0x0008	U	the mate is unmapped
0x0010	r	strand of the query (1 for reverse)
0x0020	R	strand of the mate
0x0040	1	the read is the first read in a pair
0x0080	2	the read is the second read in a pair
0x0100	s	the alignment is not primary
0x0200	f	the read fails platform/vendor quality checks
0x0400	d	the read is either a PCR or an optical duplicate
.TE

.SH EXAMPLES
.IP o 2
Import SAM to BAM when
.B @SQ
lines are present in the header:

  samtools view -bS aln.sam > aln.bam

If
.B @SQ
lines are absent:

  samtools faidx ref.fa
  samtools view -bt ref.fa.fai aln.sam > aln.bam

where
.I ref.fa.fai
is generated automatically by the
.B faidx
command.

.IP o 2
Attach the
.B RG
tag while merging sorted alignments:

  perl -e 'print "@RG\\tID:ga\\tSM:hs\\tLB:ga\\tPL:Illumina\\n@RG\\tID:454\\tSM:hs\\tLB:454\\tPL:454\\n"' > rg.txt
  samtools merge -rh rg.txt merged.bam ga.bam 454.bam

The value in a
.B RG
tag is determined by the file name the read is coming from. In this
example, in the
.IR merged.bam ,
reads from
.I ga.bam
will be attached 
.IR RG:Z:ga ,
while reads from
.I 454.bam
will be attached
.IR RG:Z:454 .

.IP o 2
Call SNPs and short indels for one diploid individual:

  samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
  bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 > var.flt.vcf

The
.B -D
option of varFilter controls the maximum read depth, which should be
adjusted to about twice the average read depth.  One may consider to add
.B -C50
to
.B mpileup
if mapping quality is overestimated for reads containing excessive
mismatches. Applying this option usually helps
.B BWA-short
but may not other mappers.

.IP o 2
Generate the consensus sequence for one diploid individual:

  samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq

.IP o 2
Phase one individual:

  samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out

The
.B calmd
command is used to reduce false heterozygotes around INDELs.

.IP o 2
Call SNPs and short indels for multiple diploid individuals:

  samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf
  bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf

Individuals are identified from the
.B SM
tags in the
.B @RG
header lines. Individuals can be pooled in one alignment file; one
individual can also be separated into multiple files. The
.B -P
option specifies that indel candidates should be collected only from
read groups with the
.B @RG-PL
tag set to
.IR ILLUMINA .
Collecting indel candidates from reads sequenced by an indel-prone
technology may affect the performance of indel calling.

.IP o 2
Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals:

  samtools mpileup -Igf ref.fa *.bam > all.bcf
  bcftools view -bl sites.list all.bcf > sites.bcf
  bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
  bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
  bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
  ......

where
.I sites.list
contains the list of sites with each line consisting of the reference
sequence name and position. The following
.B bcftools
commands estimate AFS by EM.

.IP o 2
Dump BAQ applied alignment for other SNP callers:

  samtools calmd -bAr aln.bam > aln.baq.bam

It adds and corrects the
.B NM
and
.B MD
tags at the same time. The
.B calmd
command also comes with the
.B -C
option, the same as the one in
.B pileup
and
.BR mpileup .
Apply if it helps.

.SH LIMITATIONS
.PP
.IP o 2
Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
.IP o 2
In merging, the input files are required to have the same number of
reference sequences. The requirement can be relaxed. In addition,
merging does not reconstruct the header dictionaries
automatically. Endusers have to provide the correct header. Picard is
better at merging.
.IP o 2
Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan
reads or ends mapped to different chromosomes). If this is a concern,
please use Picard's MarkDuplicate which correctly handles these cases,
although a little slower.

.SH AUTHOR
.PP
Heng Li from the Sanger Institute wrote the C version of samtools. Bob
Handsaker from the Broad Institute implemented the BGZF library and Jue
Ruan from Beijing Genomics Institute wrote the RAZF library. John
Marshall and Petr Danecek contribute to the source code and various
people from the 1000 Genomes Project have contributed to the SAM format
specification.

.SH SEE ALSO
.PP
Samtools website: <http://samtools.sourceforge.net>