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.TH samtools 1 "2 September 2009" "samtools-0.1.6" "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 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 -f ref.fasta aln.sorted.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 import
samtools import <in.ref_list> <in.sam> <out.bam>

Since 0.1.4, this command is an alias of:

samtools view -bt <in.ref_list> -o <out.bam> <in.sam>

.TP
.B sort
samtools sort [-n] [-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 -n
Sort by read names rather than by chromosomal coordinates
.TP
.B -m INT
Approximately the maximum required memory. [500000000]
.RE

.TP
.B merge
samtools merge [-h inh.sam] [-n] <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
.B -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
.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 view
samtools view [-bhuHS] [-t in.refList] [-o output] [-f reqFlag] [-F
skipFlag] [-q minMapQ] [-l library] [-r readGroup] <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', `chr2:1000000' or `chr2:1,000,000-2,000,000'. The
coordinate is 1-based.

.B OPTIONS:
.RS
.TP 8
.B -b
Output in the BAM format.
.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.
.TP
.B -h
Include the header in the output.
.TP
.B -H
Output the header only.
.TP
.B -S
Input is in SAM. If @SQ header lines are absent, the
.B `-t'
option is required.
.TP
.B -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 -o FILE
Output file [stdout]
.TP
.B -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
.B -F INT
Skip alignments with bits present in INT [0]
.TP
.B -q INT
Skip alignments with MAPQ smaller than INT [0]
.TP
.B -l STR
Only output reads in library STR [null]
.TP
.B -r STR
Only output reads in read group STR [null]
.RE

.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 pileup
samtools pileup [-f in.ref.fasta] [-t in.ref_list] [-l in.site_list]
[-iscgS2] [-T theta] [-N nHap] [-r pairDiffRate] <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,
`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 segment
which is a contiguous subsequence on the read separated by `N/S/H' CIGAR
operations. 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, consensus quality, SNP quality and 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 OPTIONS:
.RS

.TP 10
.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
.B -i
Only output pileup lines containing indels.

.TP
.B -f FILE
The reference sequence in the FASTA format. Index file
.I FILE.fai
will be created if
absent.

.TP
.B -M INT
Cap mapping quality at INT [60]

.TP
.B -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.

.TP
.B -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
.B -s
together with
.B -l
as in the default format we may not know the mapping quality.

.TP
.B -c
Call the consensus sequence using MAQ consensus model. Options
.B -T,
.B -N,
.B -I
and
.B -r
are only effective when
.B -c
or
.B -g
is in use.

.TP
.B -g
Generate genotype likelihood in the binary GLFv3 format. This option
suppresses -c, -i and -s.

.TP
.B -T FLOAT
The theta parameter (error dependency coefficient) in the maq consensus
calling model [0.85]

.TP
.B -N INT
Number of haplotypes in the sample (>=2) [2]

.TP
.B -r FLOAT
Expected fraction of differences between a pair of haplotypes [0.001]

.TP
.B -I INT
Phred probability of an indel in sequencing/prep. [40]

.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'.

.RE

.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 <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.
This command
.B ONLY
works with FR orientation and requires ISIZE is correctly set.

.RE

.TP
.B rmdupse
samtools rmdupse <input.srt.bam> <out.bam>

Remove potential duplicates for single-ended reads. This command will
treat all reads as single-ended even if they are paired in fact.

.RE

.TP
.B fillmd
samtools fillmd [-e] <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.

.B OPTIONS:
.RS
.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.

.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
l | l .
Flag	Description
_
0x0001	the read is paired in sequencing
0x0002	the read is mapped in a proper pair
0x0004	the query sequence itself is unmapped
0x0008	the mate is unmapped
0x0010	strand of the query (1 for reverse)
0x0020	strand of the mate
0x0040	the read is the first read in a pair
0x0080	the read is the second read in a pair
0x0100	the alignment is not primary
0x0200	the read fails platform/vendor quality checks
0x0400	the read is either a PCR or an optical duplicate
.TE

.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
CIGAR operation P is not properly handled at the moment.
.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' rmdup does not work for single-end data and does not remove
duplicates across chromosomes. Picard is better.

.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. Various
people in the 1000Genomes Project contributed to the SAM format
specification.

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