samtools.1

.TH samtools 1 "15 August 2014" "samtools-1.0" "Bioinformatics tools"
.SH NAME
samtools \- Utilities for the Sequence Alignment/Map (SAM) format
.\"
.\" Copyright (C) 2008-2011, 2013-2014 Genome Research Ltd.
.\" Portions copyright (C) 2010, 2011 Broad Institute.
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.\" Author: Heng Li <lh3@sanger.ac.uk>
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.SH SYNOPSIS
.PP
samtools view -bt ref_list.txt -o aln.bam aln.sam.gz
.PP
samtools sort -T /tmp/aln.sorted -o aln.sorted.bam aln.bam
.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 mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
.PP
samtools tview aln.sorted.bam ref.fasta
.PP
samtools flags PAIRED,UNMAP,MUNMAP
.PP
samtools bam2fq input.bam > output.fastq

.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
.RI [ options ]
.IR in.bam | in.sam | in.cram
.RI [ region ...]

With no options or regions specified, prints all alignments in the specified
input alignment file (in SAM, BAM, or CRAM format) to standard output
in SAM format (with no header).

You may specify one or more space-separated region specifications after the
input filename to restrict output to only those alignments which overlap the
specified region(s). Use of region specifications requires a coordinate-sorted
and indexed input file (in BAM or CRAM format).

The
.BR -b ,
.BR -C ,
.BR -1 ,
.BR -u ,
.BR -h ,
.BR -H ,
and
.B -c
options change the output format from the default of headerless SAM, and the
.B -o
and
.B -U
options set the output file name(s).

The
.B -t
and
.B -T
options provide additional reference data. One of these two options is required
when SAM input does not contain @SQ headers, and the
.B -T
option is required whenever writing CRAM output.

The
.BR -L ,
.BR -r ,
.BR -R ,
.BR -q ,
.BR -l ,
.BR -m ,
.BR -f ,
and
.B -F
options filter the alignments that will be included in the output to only those
alignments that match certain criteria.

The
.BR -x ,
.BR -B ,
.BR -s ,
and
.B -Q
options modify the data which is contained in each alignment.

Finally, the
.B -@
option can be used to allocate additional threads to be used for compression, and the
.B -?
option requests a long help message.

.TP
.B REGIONS:
.RS
Regions can be specified as: RNAME[:STARTPOS[-ENDPOS]] and all position
coordinates are 1-based.

Important note: when multiple regions are given, some alignments may be output
multiple times if they overlap more than one of the specified regions.

Examples of region specifications:
.TP 10
`chr1'
Output all alignments mapped to the reference sequence named `chr1' (i.e. @SQ SN:chr1) .
.TP
`chr2:1000000'
The region on chr2 beginning at base position 1,000,000 and ending at the
end of the chromosome.
.TP
`chr3:1000-2000'
The 1001bp region on chr3 beginning at base position 1,000 and ending at base
position 2,000 (including both end positions).
.RE

.B OPTIONS:
.RS
.TP 10
.B -b
Output in the BAM format.
.TP
.B -C
Output in the CRAM format (requires -T).
.TP
.B -1
Enable fast BAM compression (implies -b).
.TP
.B -u
Output uncompressed BAM. This option saves time spent on
compression/decompression and is thus preferred when the output is piped
to another samtools command.
.TP
.B -h
Include the header in the output.
.TP
.B -H
Output the header only.
.TP
.B -c
Instead of printing the alignments, only count them and print the
total number. All filter options, such as
.BR -f ,
.BR -F ,
and
.BR -q ,
are taken into account.
.TP
.B -?
Output long help and exit immediately.
.TP
.BI "-o " FILE
Output to
.I FILE [stdout].
.TP
.BI "-U " FILE
Write alignments that are
.I not
selected by the various filter options to
.IR FILE .
When this option is used, all alignments (or all alignments intersecting the
.I regions
specified) are written to either the output file or this file, but never both.
.TP
.BI "-t " FILE
A tab-delimited
.IR FILE .
Each line must contain the reference name in the first column and the length of
the reference in the second column, with one line for each distinct reference.
Any additional fields beyond the second column are ignored. This file also
defines the order of the reference sequences in sorting. If you run:
`samtools faidx <ref.fa>', the resulting index file
.I <ref.fa>.fai
can be used as this
.IR FILE .
.TP
.BI "-T " FILE
A FASTA format reference
.IR FILE ,
optionally compressed by
.B bgzip
and ideally indexed by
.B samtools
.BR faidx .
If an index is not present, one will be generated for you.
.TP
.BI "-L " FILE
Only output alignments overlapping the input BED
.I FILE
[null].
.TP
.BI "-r " STR
Only output alignments in read group
.I STR
[null].
.TP
.BI "-R " FILE
Output alignments in read groups listed in
.I FILE
[null].
.TP
.BI "-q " INT
Skip alignments with MAPQ smaller than
.I INT
[0].
.TP
.BI "-l " STR
Only output alignments in library
.I STR
[null].
.TP
.BI "-m " INT
Only output alignments with number of CIGAR bases consuming query
sequence \(>=
.I INT
[0]
.TP
.BI "-f " INT
Only output alignments with all bits set in
.I INT
present in the FLAG field.
.I INT
can be specified in hex by beginning with `0x' (i.e. /^0x[0-9A-F]+/)
or in octal by beginning with `0' (i.e. /^0[0-7]+/) [0].
.TP
.BI "-F " INT
Do not output alignments with any bits set in
.I INT
present in the FLAG field.
.I INT
can be specified in hex by beginning with `0x' (i.e. /^0x[0-9A-F]+/)
or in octal by beginning with `0' (i.e. /^0[0-7]+/) [0].
.TP
.BI "-x " STR
Read tag to exclude from output (repeatable) [null]
.TP
.B -B
Collapse the backward CIGAR operation.
.TP
.BI "-s " FLOAT
Integer part is used to seed the random number generator [0]. Part after the
decimal point sets the fraction of templates/pairs to subsample [no subsampling].
.TP
.BI "-Q " INT
Scale quality values in output by INT [1]
.TP
.BI "-@ " INT
Number of BAM compression threads to use in addition to main thread [0].
.TP
.B -S
Ignored for compatibility with previous samtools versions.
Previously this option was required if input was in SAM format, but now the
correct format is automatically detected by examining the first few characters
of input.
.RE

.TP
.B tview
samtools tview
.RB [ \-p
.IR chr:pos ]
.RB [ \-s
.IR STR ]
.RB [ \-d
.IR display ]
.RI <in.sorted.bam>
.RI [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.

.B Options:
.RS
.TP 14
.BI -d \ display
Output as (H)tml or (C)urses or (T)ext
.TP
.BI -p \ chr:pos
Go directly to this position
.TP
.BI -s \ STR
Display only alignments from this sample or read group
.RE

.TP
.B mpileup
samtools mpileup
.RB [ \-EBugp ]
.RB [ \-C
.IR capQcoef ]
.RB [ \-r
.IR reg ]
.RB [ \-f
.IR in.fa ]
.RB [ \-l
.IR list ]
.RB [ \-Q
.IR minBaseQ ]
.RB [ \-q
.IR minMapQ ]
.I in.bam
.RI [ in2.bam
.RI [ ... ]]

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

In the pileup format (without
.BR -u \ or \ -g ),
each
line represents a genomic position, consisting of chromosome name,
1-based coordinate, reference base, the number of reads covering the site,
read bases, base 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.

.B Input Options:
.RS
.TP 10
.B -6, --illumina1.3+
Assume the quality is in the Illumina 1.3+ encoding.
.TP
.B -A, --count-orphans
Do not skip anomalous read pairs in variant calling.
.TP
.BI -b,\ --bam-list \ FILE
List of input BAM files, one file per line [null]
.TP
.B -B, --no-BAQ
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,\ --adjust-MQ \ 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 -d,\ --max-depth \ INT
At a position, read maximally
.I INT
reads per input BAM. [250]
.TP
.B -E, --redo-BAQ
Recalculate BAQ on the fly, ignore existing BQ tags
.TP
.BI -f,\ --fasta-ref \ FILE
The
.BR faidx -indexed
reference file in the FASTA format. The file can be optionally compressed by
.BR bgzip .
[null]
.TP
.BI -G,\ --exclude-RG \ FILE
Exclude reads from readgroups listed in FILE (one @RG-ID per line)
.TP
.BI -l,\ --positions \ FILE
BED or position list file containing a list of regions or sites where
pileup or BCF should be generated. If BED, positions are 0-based
half-open [null]
.TP
.BI -q,\ -min-MQ \ INT
Minimum mapping quality for an alignment to be used [0]
.TP
.BI -Q,\ --min-BQ \ INT
Minimum base quality for a base to be considered [13]
.TP
.BI -r,\ --region \ STR
Only generate pileup in region. Requires the BAM files to be indexed.
If used in conjunction with -l then considers the intersection of the
two requests.
.I STR
[all sites]
.TP
.B -R,\ --ignore-RG
Ignore RG tags. Treat all reads in one BAM as one sample.
.TP
.BI --rf,\ --incl-flags \ STR|INT
Required flags: skip reads with mask bits unset [null]
.TP
.BI --ff,\ --excl-flags \ STR|INT
Filter flags: skip reads with mask bits set
[UNMAP,SECONDARY,QCFAIL,DUP]
.TP
.B -x,\ --ignore-overlaps
Disable read-pair overlap detection.
.PP
.B Output Options:
.TP 10
.BI "-o, --output " FILE
Write pileup or VCF/BCF output to
.IR FILE ,
rather than the default of standard output.

(The same short option is used for both
.B --open-prob
and
.BR --output .
If
.BR -o 's
argument contains any non-digit characters other than a leading + or - sign,
it is interpreted as
.BR --output .
Usually the filename extension will take care of this, but to write to an
entirely numeric filename use
.B -o ./123
or
.BR "--output 123" .)
.TP
.B -g,\ --BCF
Compute genotype likelihoods and output them in the binary call format (BCF).
As of v1.0, this is BCF2 which is incompatible with the BCF1 format produced
by previous (0.1.x) versions of samtools.
.TP
.B -v,\ --VCF
Compute genotype likelihoods and output them in the variant call format (VCF).
Output is bgzip-compressed VCF unless
.B -u
option is set.
.PP
.B Output Options for mpileup format (without -g or -v):
.TP 10
.B -O, --output-BP
Output base positions on reads.
.TP
.B -s, --output-MQ
Output mapping quality.
.PP
.B Output Options for VCF/BCF format (with -g or -v):
.TP 10
.B -D
Output per-sample read depth [DEPRECATED - use
.B -t DP
instead]
.TP
.B -S
Output per-sample Phred-scaled strand bias P-value [DEPRECATED - use
.B -t SP
instead]
.TP
.BI -t,\ --output-tags \ LIST
Comma-separated list of FORMAT and INFO tags to output (case-insensitive):
.B DP
(Number of high-quality bases, FORMAT),
.B DV
(Number of high-quality non-reference bases, FORMAT),
.B DPR
(Number of high-quality bases for each observed allele, FORMAT),
.B INFO/DPR
(Number of high-quality bases for each observed allele, INFO),
.B DP4
(Number of high-quality ref-forward, ref-reverse, alt-forward and alt-reverse bases, FORMAT),
.B SP
(Phred-scaled strand bias P-value, FORMAT)
[null]
.TP
.B -u,\ --uncompressed
Generate uncompressed VCF/BCF output, which is preferred for piping.
.TP
.B -V
Output per-sample number of non-reference reads [DEPRECATED - use
.B -t DV
instead]
.PP
.B Options for SNP/INDEL Genotype Likelihood Computation (for -g or -v):
.TP 10
.BI -e,\ --ext-prob \ INT
Phred-scaled gap extension sequencing error probability. Reducing
.I INT
leads to longer indels. [20]
.TP
.BI -F,\ --gap-frac \ FLOAT
Minimum fraction of gapped reads [0.002]
.TP
.BI -h,\ --tandem-qual \ 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
.B -I, --skip-indels
Do not perform INDEL calling
.TP
.BI -L,\ --max-idepth \ INT
Skip INDEL calling if the average per-sample depth is above
.IR INT .
[250]
.TP
.BI -m,\ --min-ireads \ INT
Minimum number gapped reads for indel candidates
.IR INT .
[1]
.TP
.BI -o,\ --open-prob \ INT
Phred-scaled gap open sequencing error probability. Reducing
.I INT
leads to more indel calls. [40]

(The same short option is used for both
.B --open-prob
and
.BR --output .
When
.BR -o 's
argument contains only an optional + or - sign followed by the digits 0 to 9,
it is interpreted as
.BR --open-prob .)
.TP
.B -p, --per-sample-mF
Apply
.B -m
and
.B -F
thresholds per sample to increase sensitivity of calling.
By default both options are applied to reads pooled from all samples.
.TP
.BI -P,\ --platforms \ STR
Comma-delimited 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]
.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 cat
samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ]

Concatenate BAMs. The sequence dictionary of each input BAM must be identical,
although this command does not check this. This command uses a similar trick
to
.B reheader
which enables fast BAM concatenation.

.TP
.B sort
.na
samtools sort
.RB [ -l
.IR level ]
.RB [ -m
.IR maxMem ]
.RB [ -o
.IR out.bam ]
.RB [ -O
.IR format ]
.RB [ -n ]
.BI "-T " out.prefix
.RB [ -@
.IR threads "] [" in.bam ]
.ad

Sort alignments by leftmost coordinates, or by read name when
.B -n
is used.
An appropriate
.B @HD-SO
sort order header tag will be added or an existing one updated if necessary.

The sorted output is written to standard output by default, or to the
specified file
.RI ( out.bam )
when
.B -o
is used.
This command will also create temporary files
.IB out.prefix . %d .bam
as needed when the entire alignment data cannot fit into memory
(as controlled via the
.B -m
option).

.B Options:
.RS
.TP 11
.BI "-l " INT
Set the compression level, from 0 to 9, for the final output file.
If
.B -l
is not used, the default compression level will apply.
.TP
.BI "-m " INT
Approximately the maximum required memory per thread, specified either in bytes
or with a
.BR K ", " M ", or " G
suffix.
[768 MiB]
.TP
.B -n
Sort by read names (i.e., the
.B QNAME
field) rather than by chromosomal coordinates.
.TP
.BI "-o " FILE
Write the final sorted output to
.IR FILE ,
rather than to standard output.
.TP
.BI "-O " FORMAT
Write the final output as
.BR sam ", " bam ", or " cram .

By default, samtools tries to select a format based on the
.B -o
filename extension; if output is to standard output or no format can be
deduced,
.B -O
must be used.
.TP
.BI "-T " PREFIX
Write temporary files to
.IB PREFIX . nnnn .bam.
This option is required.
.TP
.BI "-@ " INT
Set number of sorting and compression threads.
By default, operation is single-threaded.
.PP
For compatibility with existing scripts,
.B samtools sort
also accepts the previous less flexible way of specifying the final and
temporary output filenames:
.PP
samtools sort
.RB [ -nof "] [" -m
.IR maxMem ]
.I in.bam out.prefix

The sorted BAM output is written to
.IB out.prefix .bam
(or as determined by the
.B -o
and
.B -f
options below) and any temporary files are written alongside as
.IB out.prefix . %d .bam.

.TP 5
.B -o
Output the final alignment to the standard output.
.TP
.B -f
Use
.I out.prefix
as the full output path and do not append
.B .bam
suffix.
.TP
.BR -l ", " -m ", " -n ", " -@
Accepted with the same meanings as above.
.PP
This will eventually be removed; you should move to using the more flexible
newer style of invocation.
.RE

.TP
.B merge
samtools merge [-nur1f] [-h inh.sam] [-R reg] [-b <list>] <out.bam> <in1.bam> <in2.bam> [<in3.bam> ... <inN.bam>]

Merge multiple sorted alignment files, producing a single sorted output file
that contains all the input records and maintains the existing sort order.

If
.BR -h
is specified the @SQ headers of input files will be merged into the specified header, otherwise they will be merged
into a composite header created from the input headers.  If in the process of merging @SQ lines for coordinate sorted
input files, a conflict arises as to the order (for example input1.bam has @SQ for a,b,c and input2.bam has b,a,c)
then the resulting output file will need to be re-sorted back into coordinate order.

Unless the
.BR -c
or
.BR -p
flags are specified then when merging @RG and @PG records into the output header then any IDs found to be duplicates
of existing IDs in the output header will have a suffix appended to them to diffientiate them from similar header
records from other files and the read records will be updated to reflect this.

.B OPTIONS:
.RS
.TP 8
.B -1
Use zlib compression level 1 to compress the output.
.TP
.BI -b \ FILE
List of input BAM files, one file per line.
.TP
.B -f
Force to overwrite the output file if present.
.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
.B -n
The input alignments are sorted by read names rather than by chromosomal
coordinates
.TP
.BI -R \ STR
Merge files in the specified region indicated by
.I STR
[null]
.TP
.B -r
Attach an RG tag to each alignment. The tag value is inferred from file names.
.TP
.B -u
Uncompressed BAM output
.TP
.B -c
Combine RG tags with colliding IDs rather than adding a suffix to differentiate them.
.TP
.B -p
Combine PG tags with colliding IDs rather than adding a suffix to differentiate them.
.RE

.TP
.B index
samtools index
.RB [ -bc ]
.RB [ -m
.IR INT ]
.IR aln.bam | aln.cram

Index a coordinate-sorted BAM or CRAM file for fast random access.
This index is needed when
.I region
arguments are used to limit
.B samtools view
and similar commands to particular regions of interest.

For a CRAM file
.IR aln.cram ,
index file
.IB aln.cram .crai
will be created.
For a BAM file
.IR aln.bam ,
either
.IB aln.bam .bai
or
.IB aln.bam .csi
will be created, depending on the index format selected.

.B Options:
.RS
.TP 8
.B -b
Create a BAI index.
This is currently the default when no format options are used.
.TP
.B -c
Create a CSI index.
By default, the minimum interval size for the index is 2^14, which is the same
as the fixed value used by the BAI format.
.TP
.BI "-m " INT
Create a CSI index, with a minimum interval size of 2^INT.
.RE

.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 specified, the subsequences will be
retrieved and printed to stdout in the FASTA format. The input file can
be compressed in the
.B BGZF
format.

.TP
.B fixmate
samtools fixmate [-rp] <in.nameSrt.bam> <out.bam>

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

.B OPTIONS:
.RS
.TP 8
.B -r
Remove secondary and unmapped reads.
.TP 8
.B -p
Disable FR proper pair check.
.RE

.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 duplicates 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)].

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

Call and phase heterozygous SNPs.

.B OPTIONS:
.RS
.TP 8
.B -A
Drop reads with ambiguous phase.
.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 flags
samtools flags INT|STR[,...]

Convert between textual and numeric flag representation.

.B FLAGS:
.TS
tab(%);
l l l .
0x1 % PAIRED % .. paired-end (or multiple-segment) sequencing technology
0x2 % PROPER_PAIR % .. each segment properly aligned according to the aligner
0x4 % UNMAP % .. segment unmapped
0x8 % MUNMAP % .. next segment in the template unmapped
0x10 % REVERSE % .. SEQ is reverse complemented
0x20 % MREVERSE % .. SEQ of the next segment in the template is reversed
0x40 % READ1 % .. the first segment in the template
0x80 % READ2 % .. the last segment in the template
0x100 % SECONDARY % .. secondary alignment
0x200 % QCFAIL % .. not passing quality controls
0x400 % DUP % .. PCR or optical duplicate
0x800 % SUPPLEMENTARY % .. supplementary alignment
.TE

.TP
.B bam2fq
samtools bam2fq [-nO] [-s <outSE.fq>] <in.bam>

Converts a bam into FASTQ format.

.B OPTIONS:
.RS
.TP 8
.B -n
By default, either '/1' or '/2' is added to the end of read names
where the corresponding BAM_READ1 or BAM_READ2 flag is set.
Using
.B -n
causes read names to be left as they are.
.TP 8
.B -O
Use quality values from OQ tags in preference to standard quality string
if available.
.TP 8
.B -s FILE
Write singleton reads in FASTQ format to FILE instead of outputting them.
.RE


.TP
.BR help ,\  --help
Display a brief usage message listing the samtools commands available.
If the name of a command is also given, e.g.,
.BR samtools\ help\ view ,
the detailed usage message for that particular command is displayed.

.TP
.B --version
Display the version numbers and copyright information for samtools and
the important libraries used by samtools.

.TP
.B --version-only
Display the full samtools version number in a machine-readable format.
.PP
.SH REFERENCE SEQUENCES
.PP
The CRAM format requires use of a reference sequence for both reading
and writing.
.PP
When reading a CRAM the \fB@SQ\fR headers are interrogated to identify
the reference sequence MD5sum (\fBM5:\fR tag) and the local reference
sequence filename (\fBUR:\fR tag).  Note that \fIhttp://\fR and
\fIftp://\fR based URLs in the UR: field are not used, but local fasta
filenames (with or without \fIfile://\fR) can be used.
.PP
To create a CRAM the \fB@SQ\fR headers will also be read to identify
the reference sequences, but M5: and UR: tags may not be present. In
this case the \fB-T\fR and \fB-t\fR options of samtools view may be
used to specify the fasta or fasta.fai filenames respectively
(provided the .fasta.fai file is also backed up by a .fasta file).
.PP
The search order to obtain a reference is:
.IP 1. 3
Use any local file specified by the command line options (eg -T).
.IP 2. 3
Look for MD5 via REF_CACHE environment variable.
.IP 3. 3
Look for MD5 in each element of the REF_PATH environment variable.
.IP 4. 3
Look for a local file listed in the UR: header tag.
.PP
.SH ENVIRONMENT VARIABLES
.PP
.TP
.B REF_PATH
A colon separated (semi-colon on Windows) list of locations in which
to look for sequences identified by their MD5sums.  This can be either
a list of directories or URLs. Note that if a URL is included then the
colon in http:// and ftp:// and the optional port number will be
treated as part of the URL and not a PATH field separator.

For URLs, the text \fB%s\fR will be replaced by the MD5sum being
read. If no REF_PATH has been specified it will default to
\fBhttp://www.ebi.ac.uk/ena/cram/md5/%s\fR

.TP
.B REF_CACHE
This can be defined to a single directory housing a local cache of
references.  Upon downloading a reference it will be stored in the
location pointed to by REF_CACHE.  When reading a reference it will be
looked for in this directory before searching REF_PATH.  To avoid many
files being stored in the same directory, a pathname may be
constructed using %\fInum\fRs and %s notation, consuming \fInum\fR
characters of the MD5sum.  For example
\fB/local/ref_cache/%2s/%2s/%s\fR will create 2 nested subdirectories
with the filenames in the deepest directory being the last 28
characters of the md5sum.

The REF_CACHE directory will be searched for before attempting to load
via the REF_PATH search list.

To aid population of the REF_CACHE directory a script
\fBmisc/seq_ref_populate.pl\fR is provided in the Samtools
distribution. This takes a fasta file or a directory of fasta files
and generates the MD5sum named files.
.PP
.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
Convert a BAM file to a CRAM file using a local reference sequence.

  samtools view -C -T ref.fa aln.bam > aln.cram

.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:

  samtools mpileup -uf ref.fa aln.bam | bcftools call -mv > var.raw.vcf
  bcftools filter -s LowQual -e '%QUAL<20 || DP>100' var.raw.vcf  > var.flt.vcf

The
.B bcftools filter
command marks low quality sites and sites with the read depth exceeding
a limit, which should be adjusted to about twice the average read depth
(bigger read depths usually indicate problematic regions which are
often enriched for artefacts).  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.

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
Generate the consensus sequence for one diploid individual:

  samtools mpileup -uf ref.fa aln.bam | bcftools call -c | 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
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
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 MarkDuplicates 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. 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
.IR bcftools (1),
.IR sam (5)
.PP
Samtools website: <http://samtools.sourceforge.net>
.br
Samtools latest source: <https://github.com/samtools/samtools>
.br
File format specification of SAM/BAM,CRAM,VCF/BCF: <http://samtools.github.io/hts-specs>
.br
HTSlib website: <https://github.com/samtools/htslib>
.br
Bcftools website: <http://samtools.github.io/bcftools>