samtools

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Version: 372366 (fedora - 01/12/10)

Section: 1 (Commandes utilisateur)

NAME

samtools - Utilities for the Sequence Alignment/Map (SAM) format

SYNOPSIS

samtools view -bt ref_list.txt -o aln.bam aln.sam.gz

samtools sort aln.bam aln.sorted

samtools index aln.sorted.bam

samtools idxstats aln.sorted.bam

samtools view aln.sorted.bam chr2:20,100,000-20,200,000

samtools merge out.bam in1.bam in2.bam in3.bam

samtools faidx ref.fasta

samtools pileup -f ref.fasta aln.sorted.bam

samtools mpileup -f ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam

samtools tview aln.sorted.bam ref.fasta

DESCRIPTION

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.

COMMANDS AND OPTIONS

view
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 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.

OPTIONS:

-b
Output in the BAM format.
-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.
-h
Include the header in the output.
-H
Output the header only.
-S
Input is in SAM. If @SQ header lines are absent, the `-t' option is required.
-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 <ref.fa>.fai can be used as this <in.ref_list> file.
-o FILE
Output file [stdout]
-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]
-F INT
Skip alignments with bits present in INT [0]
-q INT
Skip alignments with MAPQ smaller than INT [0]
-l STR
Only output reads in library STR [null]
-r STR
Only output reads in read group STR [null]
-R FILE
Output reads in read groups listed in FILE [null]
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.

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 -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.

The position of indels is offset by -1.

OPTIONS:

-s
Print the mapping quality as the last column. This option makes the output easier to parse, although this format is not space efficient.
-S
The input file is in SAM.
-i
Only output pileup lines containing indels.
-f FILE
The reference sequence in the FASTA format. Index file FILE.fai will be created if absent.
-M INT
Cap mapping quality at INT [60]
-m INT
Filter reads with flag containing bits in INT [1796]
-d INT
Use the first NUM reads in the pileup for indel calling for speed up. Zero for unlimited. [0]
-t FILE
List of reference names ane sequence lengths, in the format described for the import command. If this option is present, samtools assumes the input <in.alignment> is in SAM format; otherwise it assumes in BAM format.
-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 -s together with -l as in the default format we may not know the mapping quality.
-c
Call the consensus sequence using SOAPsnp consensus model. Options -T, -N, -I and -r are only effective when -c or -g is in use.
-g
Generate genotype likelihood in the binary GLFv3 format. This option suppresses -c, -i and -s.
-T FLOAT
The theta parameter (error dependency coefficient) in the maq consensus calling model [0.85]
-N INT
Number of haplotypes in the sample (>=2) [2]
-r FLOAT
Expected fraction of differences between a pair of haplotypes [0.001]
-I INT
Phred probability of an indel in sequencing/prep. [40]
mpileup
samtools mpileup [-r reg] [-f in.fa] in.bam [in2.bam [...]]

Generate pileup for multiple BAM files. Consensus calling is not implemented.

OPTIONS:

-r STR
Only generate pileup in region STR [all sites]
-f FILE
The reference file [null]
reheader
samtools reheader <in.header.sam> <in.bam>

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

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

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

OPTIONS:

-o
Output the final alignment to the standard output.
-n
Sort by read names rather than by chromosomal coordinates
-m INT
Approximately the maximum required memory. [500000000]
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 inh.sam, if any, must all refer to the same set of reference sequences. The header reference list and (unless overridden by -h) `@' headers of in1.bam will be copied to out.bam, and the headers of other files will be ignored.

OPTIONS:

-h FILE
Use the lines of FILE as `@' headers to be copied to out.bam, replacing any header lines that would otherwise be copied from in1.bam. (FILE is actually in SAM format, though any alignment records it may contain are ignored.)
-r
Attach an RG tag to each alignment. The tag value is inferred from file names.
-n
The input alignments are sorted by read names rather than by chromosomal coordinates
index
samtools index <aln.bam>

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

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.

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, faidx will index the file and create <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 RAZF format.

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

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

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 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).

OPTIONS:

-s
Remove duplicate for single-end reads. By default, the command works for paired-end reads only.
-S
Treat paired-end reads and single-end reads.
calmd
samtools calmd [-eubS] <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.

OPTIONS:

-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.
-u
Output uncompressed BAM
-b
Output compressed BAM
-S
The input is SAM with header lines

SAM FORMAT

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

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

Each bit in the FLAG field is defined as:

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

LIMITATIONS

o
Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
o
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.
o
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.

AUTHOR

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 1000 Genomes Project contributed to the SAM format specification.

SEE ALSO

Samtools website: <http://samtools.sourceforge.net>