samtools(1)

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 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 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

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>

sort
    samtools sort [-n] [-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:

    -n  Sort by read names rather than by chromosomal coordinates

    -m INT
        Approximately the maximum required memory. [500000000]

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 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.)

    -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.

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' (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]

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.

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. 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]

    -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 MAQ 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]

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'.

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

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.

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. 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.

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 | 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

LIMITATIONS

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

o   CIGAR operation P is not properly handled at the moment.

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' rmdup does not work for single-end data and does not remove duplicates across chromosomes. Picard is better.

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 1000Genomes Project contributed to the SAM format specification.

SEE ALSO

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