-.TH samtools 1 "28 January 2009" "samtools-0.1.2" "Bioinformatics tools"
+.TH samtools 1 "2 October 2010" "samtools-0.1.8" "Bioinformatics tools"
.SH NAME
.PP
samtools - Utilities for the Sequence Alignment/Map (SAM) format
.SH SYNOPSIS
.PP
-samtools import ref_list.txt aln.sam.gz aln.bam
+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 pileup -f ref.fasta aln.sorted.bam
.PP
+samtools mpileup -C50 -agf 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.
+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>
-
-Convert alignments in SAM format to BAM format. File
-.I <in.ref_list>
-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. File
-.I <in.sam>
-can be optionally compressed by zlib or gzip. A single hyphen is
-recognized as stdin or stdout, depending on the context. 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 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 [-n] <out.bam> <in1.bam> <in2.bam> [...]
-
-Merge multiple sorted alignments. The header of
-.I <in1.bam>
-will be copied to
-.I <out.bam>
-and the headers of other files will be ignored.
-
-.B OPTIONS:
-.RS
-.TP 8
-.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
+.TP 10
.B view
-samtools view [-bhH] <in.bam> [region1 [...]]
+samtools view [-bhuHS] [-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 with the specified regions will be output. An
+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'.
+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
.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]
+.TP
+.B -R FILE
+Output reads in read groups listed in
+.I FILE
+[null]
.RE
.TP
-.B faidx
-samtools faidx <ref.fasta> [region1 [...]]
+.B tview
+samtools tview <in.sorted.bam> [ref.fasta]
-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.
+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 pileup
samtools pileup [-f in.ref.fasta] [-t in.ref_list] [-l in.site_list]
-[-iscg] [-T theta] [-N nHap] [-r pairDiffRate] <in.alignment>
+[-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,
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. Also at
+`-[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
If option
.B -c
-is applied, the consensus base, consensus quality, SNP quality and
-maximum 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, top two high-scoring ins/del sequences, the
-number of alignments containing the first indel allele, the number of
-alignments containing the second indel allele, and the number of
-alignments containing indels different from the top two alleles.
+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.
+
+The position of indels is offset by -1.
.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 -m INT
+Filter reads with flag containing bits in
+.I
+INT
+[1796]
+.TP
+.B -d INT
+Use the first
+.I NUM
+reads in the pileup for indel calling for speed up. Zero for unlimited. [0]
.TP
.B -t FILE
List of reference names ane sequence lengths, in the format described
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
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
+Call the consensus sequence using SOAPsnp consensus model. Options
.B -T,
-.B -N
+.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 GLFv2 format. This option
+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 mpileup
+samtools mpileup [-aug] [-C coef] [-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 -a
+Perform HMM realignment to compute base alignment quality (BAQ). Base
+quality will be capped by BAQ.
+.TP
+.B -g
+Compute genotype likelihoods and output them in the binary call format (BCF).
+.TP
+.B -u
+Similar to
+.B -g
+except that the output is uncompressed BCF, which is preferred for pipeing.
+.TP
+.B -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 is 50. [0]
+.TP
+.B -f FILE
+The reference file [null]
+.TP
+.B -l FILE
+File containing a list of sites where pileup or BCF is outputted [null]
+.TP
+.B -q INT
+Minimum mapping quality for an alignment to be used [0]
+.TP
+.B -Q INT
+Minimum base quality for a base to be considered [13]
+.TP
+.B -r STR
+Only generate pileup in region
+.I STR
+[all sites]
.RE
.TP
-.B tview
-samtools tview <in.sorted.bam> [ref.fasta]
+.B reheader
+samtools reheader <in.header.sam> <in.bam>
-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'. Note that if the region
-showed on the screen contains no mapped reads, a blank screen will be
-seen. This is a known issue and will be improved later.
+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
+.B -m INT
+Approximately the maximum required memory. [500000000]
.RE
+.TP
+.B merge
+samtools merge [-h inh.sam] [-nr] <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 -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
+.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>
.TP
.B rmdup
-samtools rmdup <input.srt.bam> <out.bam>
+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.
-This command
+In the paired-end mode, this command
.B ONLY
-works with FR orientation and requires ISIZE is correctly set.
+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 [-eubS] <aln.bam> <ref.fasta>
-.SH SAM FORFAM
+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 -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
+.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
-l | l .
-Flag Description
+cb | cb | cb
+l | c | l .
+Flag Chr 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
+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 LIMITATIONS
.PP
.IP o 2
-In general, more testing is needed to ensure there is no severe bug.
-.IP o 2
-Reference sequence names and lengths are not acquired from the BAM/SAM header.
+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.
+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
-There is a small memory leak in the viewer.
+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. Various
-people in the 1000Genomes Project contributed to the SAM format
+people in the 1000 Genomes Project contributed to the SAM format
specification.
.SH SEE ALSO
.PP
-Samtools website: http://samtools.sourceforge.net
+Samtools website: <http://samtools.sourceforge.net>
projects / samtools.git / blobdiff