samtools manual(1)

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Manual Reference Pages  - samtools (1)

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NAME

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

bcftools - Utilities for the Binary Call Format (BCF) and VCF

CONTENTS

Synopsis
Description
Samtools Commands And Options
Bcftools Commands And Options
Sam Format
Vcf Format
Examples
Limitations
Author
See Also

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

bcftools index in.bcf

bcftools view in.bcf chr2:100-200 > out.vcf

bcftools view -vc in.bcf > out.vcf 2> out.afs

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.

SAMTOOLS COMMANDS AND OPTIONS

viewsamtools view [-bchuHS] [-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:-bOutput in the BAM format.-f INTOnly 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 INTSkip alignments with bits present in INT [0]-hInclude the header in the output.-HOutput the header only.-l STROnly output reads in library STR [null]-o FILEOutput file [stdout]-q INTSkip alignments with MAPQ smaller than INT [0]-r STROnly output reads in read group STR [null]-R FILEOutput reads in read groups listed in FILE [null]-SInput is in SAM. If @SQ header lines are absent, the ‘-t’ option is required.-cInstead 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 FILEThis 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.-uOutput uncompressed BAM. This option saves time spent on compression/decomprssion and is thus preferred when the output is piped to another samtools command.

tviewsamtools 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.

mpileupsamtools mpileup [-EBug] [-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. Alignment records are grouped by sample identifiers in @RG header lines. If sample identifiers are absent, each input file is regarded as one sample.

In the pileup format (without -uor-g), 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 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. The ASCII of the character following ‘^’ minus 33 gives the mapping quality. A symbol ‘$’ marks the end of a read segment.

Input Options:-6Assume the quality is in the Illumina 1.3+ encoding. -A Do not skip anomalous read pairs in variant calling.-BDisable 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.-b FILEList of input BAM files, one file per line [null]-C INTCoefficient 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]-d INTAt a position, read maximally INT reads per input BAM. [250]-EExtended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt specificity a little bit.-f FILEThe faidx-indexed reference file in the FASTA format. The file can be optionally compressed by razip. [null]-l FILEBED or position list file containing a list of regions or sites where pileup or BCF should be generated [null]-q INTMinimum mapping quality for an alignment to be used [0]-Q INTMinimum base quality for a base to be considered [13]-r STROnly generate pileup in region STR [all sites]Output Options: 

-DOutput per-sample read depth-gCompute genotype likelihoods and output them in the binary call format (BCF).-SOutput per-sample Phred-scaled strand bias P-value-uSimilar to -g except that the output is uncompressed BCF, which is preferred for piping.

Options for Genotype Likelihood Computation (for -g or -u): 

-e INTPhred-scaled gap extension sequencing error probability. Reducing INTleads to longer indels. [20]-h INTCoefficient 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]-IDo not perform INDEL calling-L INTSkip INDEL calling if the average per-sample depth is above INT. [250]-o INTPhred-scaled gap open sequencing error probability. Reducing INT leads to more indel calls. [40]-P STRComma dilimited list of platforms (determined by @RG-PL) from which indel candidates are obtained. It is recommended to collect indel candidates from sequencing technologies that have low indel error rate such as ILLUMINA. [all]

reheadersamtools 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.

catsamtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ]

Concatenate BAMs. The sequence dictionary of each input BAM must be identical, although this command does not check this. This command uses a similar trick toreheader which enables fast BAM concatenation.

sortsamtools 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:-oOutput the final alignment to the standard output.-nSort by read names rather than by chromosomal coordinates-m INTApproximately the maximum required memory. [500000000]

mergesamtools merge [-nur1f] [-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:-1Use zlib compression level 1 to comrpess the output-fForce to overwrite the output file if present.-h FILEUse 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.)-nThe input alignments are sorted by read names rather than by chromosomal coordinates-R STRMerge files in the specified region indicated by STR [null]-rAttach an RG tag to each alignment. The tag value is inferred from file names.-uUncompressed BAM output

indexsamtools index <aln.bam>

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

idxstatssamtools 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.

faidxsamtools 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.

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

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

rmdupsamtools 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:-sRemove duplicate for single-end reads. By default, the command works for paired-end reads only.-STreat paired-end reads and single-end reads.

calmdsamtools calmd [-EeubSr] [-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 different from the existing tag. Output SAM by default.

OPTIONS:-AWhen used jointly with -r this option overwrites the original base quality.-eConvert a the read base to = if it is identical to the aligned reference base. Indel caller does not support the = bases at the moment.-uOutput uncompressed BAM-bOutput compressed BAM-SThe input is SAM with header lines-C INTCoefficient to cap mapping quality of poorly mapped reads. See the pileupcommand for details. [0]-rCompute the BQ tag (without -A) or cap base quality by BAQ (with -A).-EExtended BAQ calculation. This option trades specificity for sensitivity, though the effect is minor.

targetcutsamtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam>

This command identifies target regions by examining the continuity of read depth, computes haploid consensus sequences of targets and outputs a SAM with each sequence corresponding to a target. When option -f is in use, BAQ will be applied. This command is only designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].

phasesamtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam>

Call and phase heterozygous SNPs. OPTIONS:-ADrop reads with ambiguous phase.-b STRPrefix of BAM output. When this option is in use, phase-0 reads will be saved in fileSTR.0.bam and phase-1 reads in STR.1.bam. Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads with switch errors will be saved inSTR.chimeric.bam. [null]-FDo not attempt to fix chimeric reads.-k INTMaximum length for local phasing. [13]-q INTMinimum Phred-scaled LOD to call a heterozygote. [40]-Q INTMinimum base quality to be used in het calling. [13]

BCFTOOLS COMMANDS AND OPTIONS

viewbcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s listSample] [-igapSNPratio] [-t mutRate] [-p varThres] [-P prior] [-1 nGroup1] [-d minFrac] [-UnPerm] [-X permThres] [-T trioType] in.bcf [region]

Convert between BCF and VCF, call variant candidates and estimate allele frequencies.

Input/Output Options: 
-A Retain all possible alternate alleles at variant sites. By default, the view command discards unlikely alleles.-bOutput in the BCF format. The default is VCF.-D FILESequence dictionary (list of chromosome names) for VCF->BCF conversion [null]-FIndicate PL is generated by r921 or before (ordering is different).-GSuppress all individual genotype information.-l FILEList of sites at which information are outputted [all sites]-NSkip sites where the REF field is not A/C/G/T-QOutput the QCALL likelihood format-s FILEList of samples to use. The first column in the input gives the sample names and the second gives the ploidy, which can only be 1 or 2. When the 2nd column is absent, the sample ploidy is assumed to be 2. In the output, the ordering of samples will be identical to the one in FILE. [null]-SThe input is VCF instead of BCF.-uUncompressed BCF output (force -b).Consensus/Variant Calling Options: 
-c Call variants using Bayesian inference. This option automatically invokes option -e.-d FLOATWhen -v is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0]-ePerform max-likelihood inference only, including estimating the site allele frequency, testing Hardy-Weinberg equlibrium and testing associations with LRT.-gCall per-sample genotypes at variant sites (force -c)-i FLOATRatio of INDEL-to-SNP mutation rate [0.15]-p FLOATA site is considered to be a variant if P(ref|D)<FLOAT [0.5]-P STRPrior or initial allele frequency spectrum. If STR can be full, cond2,flat or the file consisting of error output from a previous variant calling run.-t FLOATScaled muttion rate for variant calling [0.001]-T STREnable pair/trio calling. For trio calling, option -s is usually needed to be applied to configure the trio members and their ordering. In the file supplied to the option -s, the first sample must be the child, the second the father and the third the mother. The valid values of STR are ‘pair’, ‘trioauto’, ‘trioxd’ and ‘trioxs’, where ‘pair’ calls differences between two input samples, and ‘trioxd’ (‘trioxs’) specifies that the input is from the X chromosome non-PAR regions and the child is a female (male). [null]-vOutput variant sites only (force -c)Contrast Calling and Association Test Options: 
-1 INT Number of group-1 samples. This option is used for dividing the samples into two groups for contrast SNP calling or association test. When this option is in use, the following VCF INFO will be outputted: PC2, PCHI2 and QCHI2. [0]-U INTNumber of permutations for association test (effective only with -1) [0]-X FLOATOnly perform permutations for P(chi^2)<FLOAT (effective only with -U) [0.01]

indexbcftools index in.bcf

Index sorted BCF for random access.

catbcftools cat in1.bcf ["in2.bcf "[..."]]]"

Concatenate BCF files. The input files are required to be sorted and have identical samples appearing in the same order.

SAM FORMAT

Sequence Alignment/Map (SAM) format is TAB-delimited. Apart from the header lines, which are started with the ‘@’ symbol, each alignment line consists of:

ColFieldDescription1QNAMEQuery template/pair NAME2FLAGbitwise FLAG3RNAMEReference sequence NAME4POS1-based leftmost POSition/coordinate of clipped sequence5MAPQMAPping Quality (Phred-scaled)6CIAGRextended CIGAR string7MRNMMate Reference sequence NaMe (‘=’ if same as RNAME)8MPOS1-based Mate POSistion9TLENinferred Template LENgth (insert size)10SEQquery SEQuence on the same strand as the reference11QUALquery QUALity (ASCII-33 gives the Phred base quality)12+OPTvariable OPTional fields in the format TAG:VTYPE:VALUE

Each bit in the FLAG field is defined as:

FlagChrDescription0x0001pthe read is paired in sequencing0x0002Pthe read is mapped in a proper pair0x0004uthe query sequence itself is unmapped0x0008Uthe mate is unmapped0x0010rstrand of the query (1 for reverse)0x0020Rstrand of the mate0x00401the read is the first read in a pair0x00802the read is the second read in a pair0x0100sthe alignment is not primary0x0200fthe read fails platform/vendor quality checks0x0400dthe read is either a PCR or an optical duplicate

where the second column gives the string representation of the FLAG field.

VCF FORMAT

The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:

ColFieldDescription1CHROMCHROMosome name2POSthe left-most POSition of the variant3IDunique variant IDentifier4REFthe REFerence allele5ALTthe ALTernate allele(s), separated by comma6QUALvariant/reference QUALity7FILTERFILTers applied8INFOINFOrmation related to the variant, separated by semi-colon9FORMATFORMAT of the genotype fields, separated by colon (optional)10+SAMPLESAMPLE genotypes and per-sample information (optional)

The following table gives the INFO tags used by samtools and bcftools.

TagFormatDescriptionAF1doubleMax-likelihood estimate of the site allele frequency (AF) of the first ALT alleleDPintRaw read depth (without quality filtering)DP4int[4]# high-quality reference forward bases, ref reverse, alternate for and alt rev basesFQintConsensus quality. Positive: sample genotypes different; negative: otherwiseMQintRoot-Mean-Square mapping quality of covering readsPC2int[2]Phred probability of AF in group1 samples being larger (,smaller) than in group2PCHI2doublePosterior weighted chi^2 P-value between group1 and group2 samplesPV4double[4]P-value for strand bias, baseQ bias, mapQ bias and tail distance biasQCHI2intPhred-scaled PCHI2RPint# permutations yielding a smaller PCHI2CLRintPhred log ratio of genotype likelihoods with and without the trio/pair constraintUGTstringMost probable genotype configuration without the trio constraintCGTstringMost probable configuration with the trio constraint

EXAMPLES

oImport 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.

oAttach the RG tag while merging sorted alignments:

perl -e ’print "@RG\tID:ga\tSM:hs\tLB:ga\tPL:Illumina\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 coming from. In this example, in the merged.bam, reads from ga.bam will be attached RG:Z:ga, while reads from454.bam will be attached RG:Z:454.

oCall 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 usually helps BWA-short but may not other mappers.

oGenerate the consensus sequence for one diploid individual:

samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq

oCall somatic mutations from a pair of samples:

samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf

In the output INFO field, CLR gives the Phred-log ratio between the likelihood by treating the two samples independently, and the likelihood by requiring the genotype to be identical. This CLR is effectively a score measuring the confidence of somatic calls. The higher the better.

oCall de novo and somatic mutations from a family trio:

samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf

File samples.txt should consist of three lines specifying the member and order of samples (in the order of child-father-mother). Similarly, CLR gives the Phred-log likelihood ratio with and without the trio constraint. UGT shows the most likely genotype configuration without the trio constraint, and CGT gives the most likely genotype configuration satisfying the trio constraint.

oPhase one individual:

samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out

The calmd command is used to reduce false heterozygotes around INDELs.

oCall 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.

oDerive 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.

oDump 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

oUnaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.oSamtools 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 Marshall and Petr Danecek contribute to the source code and various people from the 1000 Genomes Project have contributed to the SAM format specification.

SEE ALSO

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

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samtools-0.1.17samtools (1)05 July 2011