+++ /dev/null
-samtools(1) Bioinformatics tools 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 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 -vcf ref.fasta aln.sorted.bam
-
- samtools mpileup -C50 -gf 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 [-bchuHS] [-t in.refList] [-o output] [-f
- reqFlag] [-F skipFlag] [-q minMapQ] [-l library] [-r read-
- Group] [-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.
-
- -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]
-
- -h Include the header in the output.
-
- -H Output the header only.
-
- -l STR Only output reads in library STR [null]
-
- -o FILE Output file [stdout]
-
- -q INT Skip alignments with MAPQ smaller than INT [0]
-
- -r STR Only output reads in read group STR [null]
-
- -R FILE Output reads in read groups listed in FILE [null]
-
- -S Input is in SAM. If @SQ header lines are absent, the
- `-t' option is required.
-
- -c Instead of printing the alignments, only count them
- and print the total number. All filter options, such
- as `-f', `-F' and `-q' , are taken into account.
-
- -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.
-
- -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.
-
-
- 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 align-
- ment start from a region in the format like
- `chr10:10,000,000' or `=10,000,000' when viewing the same
- reference sequence.
-
-
- mpileup samtools mpileup [-Bug] [-C capQcoef] [-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. Align-
- ment records are grouped by sample identifiers in @RG header
- lines. If sample identifiers are absent, each input file is
- regarded as one sample.
-
- OPTIONS:
-
- -B 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.
-
- -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 for
- BWA is 50. [0]
-
- -e INT Phred-scaled gap extension sequencing error probabil-
- ity. Reducing INT leads to longer indels. [20]
-
- -f FILE The reference file [null]
-
- -g Compute genotype likelihoods and output them in the
- binary call format (BCF).
-
- -h INT Coefficient for modeling homopolymer errors. Given an
- l-long homopolymer run, the sequencing error of an
- indel of size s is modeled as INT*s/l. [100]
-
- -l FILE File containing a list of sites where pileup or BCF
- is outputted [null]
-
- -o INT Phred-scaled gap open sequencing error probability.
- Reducing INT leads to more indel calls. [40]
-
- -P STR Comma dilimited list of platforms (determined by @RG-
- PL) from which indel candidates are obtained. It is
- recommended to collect indel candidates from sequenc-
- ing technologies that have low indel error rate such
- as ILLUMINA. [all]
-
- -q INT Minimum mapping quality for an alignment to be used
- [0]
-
- -Q INT Minimum base quality for a base to be considered [13]
-
- -r STR Only generate pileup in region STR [all sites]
-
- -u Similar to -g except that the output is uncompressed
- BCF, which is preferred for piping.
-
-
- 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.pre-
- fix>.bam will be created. This command may also create tempo-
- rary files <out.prefix>.%d.bam when the whole alignment can-
- not 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 coordi-
- nates
-
- -m INT Approximately the maximum required memory.
- [500000000]
-
-
- merge samtools merge [-nur] [-h inh.sam] [-R reg] <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 other-
- wise be copied from in1.bam. (FILE is actually in
- SAM format, though any alignment records it may con-
- tain are ignored.)
-
- -R STR Merge files in the specified region indicated by STR
-
- -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
-
- -u Uncompressed BAM output
-
-
- 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 sub-
- sequence 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 com-
- mand 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 [-eubSr] [-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 dif-
- ferent from the existing tag. Output SAM by default.
-
- OPTIONS:
-
- -A When used jointly with -r this option overwrites the
- original base quality.
-
- -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
-
- -C INT Coefficient to cap mapping quality of poorly mapped
- reads. See the pileup command for details. [0]
-
- -r Compute the BQ tag without changing the base quality.
-
-
- pileup samtools pileup [-2sSBicv] [-f in.ref.fasta] [-t in.ref_list]
- [-l in.site_list] [-C capMapQ] [-M maxMapQ] [-T theta] [-N
- nHap] [-r pairDiffRate] [-m mask] [-d maxIndelDepth] [-G
- indelPrior] <in.bam>|<in.sam>
-
- Print the alignment in the pileup format. In the pileup for-
- mat, 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, a '>' or '<' for a reference skip, `ACGTN' for a mis-
- match 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]+' repre-
- sents 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 map-
- ping 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 proba-
- bility 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, coordi-
- nate, 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.
-
- NOTE: Since 0.1.10, the `pileup' command is deprecated by
- `mpileup'.
-
- OPTIONS:
-
- -B Disable the BAQ computation. See the mpileup com-
- mand for details.
-
- -c Call the consensus sequence. Options -T, -N, -I and
- -r are only effective when -c or -g is in use.
-
- -C INT Coefficient for downgrading the mapping quality of
- poorly mapped reads. See the mpileup command for
- details. [0]
-
- -d INT Use the first NUM reads in the pileup for indel
- calling for speed up. Zero for unlimited. [1024]
-
- -f FILE The reference sequence in the FASTA format. Index
- file FILE.fai will be created if absent.
-
- -g Generate genotype likelihood in the binary GLFv3
- format. This option suppresses -c, -i and -s. This
- option is deprecated by the mpileup command.
-
- -i Only output pileup lines containing indels.
-
- -I INT Phred probability of an indel in sequencing/prep.
- [40]
-
- -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
-
- -m INT Filter reads with flag containing bits in INT
- [1796]
-
- -M INT Cap mapping quality at INT [60]
-
- -N INT Number of haplotypes in the sample (>=2) [2]
-
- -r FLOAT Expected fraction of differences between a pair of
- haplotypes [0.001]
-
- -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.
-
- -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. -s together with -l as in
- the default format we may not know the mapping
- quality.
-
- -T FLOAT The theta parameter (error dependency coefficient)
- in the maq consensus calling model [0.85]
-
-
-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 |
- +-------+-----+--------------------------------------------------+
-
-EXAMPLES
- o Import SAM to BAM when @SQ lines are present in the header:
-
- samtools view -bS aln.sam > aln.bam
-
- If @SQ lines are absent:
-
- samtools faidx ref.fa
- samtools view -bt ref.fa.fai aln.sam > aln.bam
-
- where ref.fa.fai is generated automatically by the faidx command.
-
-
- o Attach the RG tag while merging sorted alignments:
-
- perl -e 'print "@RG\tID:ga\tSM:hs\tLB:ga\tPL:Illu-
- mina\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 RG tag is determined by the file name the read is com-
- ing from. In this example, in the merged.bam, reads from ga.bam will
- be attached RG:Z:ga, while reads from 454.bam will be attached
- RG:Z:454.
-
-
- o Call SNPs and short indels for one diploid individual:
-
- samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - >
- var.raw.bcf
- bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 >
- var.flt.vcf
-
- The -D option of varFilter controls the maximum read depth, which
- should be adjusted to about twice the average read depth. One may
- consider to add -C50 to mpileup if mapping quality is overestimated
- for reads containing excessive mismatches. Applying this option usu-
- ally helps BWA-short but may not other mappers.
-
-
- o Call SNPs and short indels for multiple diploid individuals:
-
- samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view
- -bcvg - > var.raw.bcf
- bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 >
- var.flt.vcf
-
- Individuals are identified from the SM tags in the @RG header lines.
- Individuals can be pooled in one alignment file; one individual can
- also be separated into multiple files. The -P option specifies that
- indel candidates should be collected only from read groups with the
- @RG-PL tag set to ILLUMINA. Collecting indel candidates from reads
- sequenced by an indel-prone technology may affect the performance of
- indel calling.
-
-
- o Derive the allele frequency spectrum (AFS) on a list of sites from
- multiple individuals:
-
- samtools mpileup -Igf ref.fa *.bam > all.bcf
- bcftools view -bl sites.list all.bcf > sites.bcf
- bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
- bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
- bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
- ......
-
- where sites.list contains the list of sites with each line consisting
- of the reference sequence name and position. The following bcftools
- commands estimate AFS by EM.
-
-
- o Dump BAQ applied alignment for other SNP callers:
-
- samtools calmd -bAr aln.bam > aln.baq.bam
-
- It adds and corrects the NM and MD tags at the same time. The calmd
- command also comes with the -C option, the same as the one in pileup
- and mpileup. Apply if it helps.
-
-
-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. John Mar-
- shall and Petr Danecek contribute to the source code and various people
- from the 1000 Genomes Project have contributed to the SAM format speci-
- fication.
-
-
-SEE ALSO
- Samtools website: <http://samtools.sourceforge.net>
-
-
-
-samtools-0.1.12 2 December 2010 samtools(1)
projects / samtools.git / blobdiff