Provided by: samtools_0.1.18-4_i386
samtools - Utilities for the Sequence Alignment/Map (SAM) format
bcftools - Utilities for the Binary Call Format (BCF) and VCF
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
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
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
view samtools 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
-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
-F INT Skip alignments with bits present in INT 
-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 
-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
alignment start from a region in the format like
`chr10:10,000,000' or `=10,000,000' when viewing the same
mpileup samtools mpileup [-EBug] [-C capQcoef] [-r reg] [-f in.fa]
[-l list] [-M capMapQ] [-Q minBaseQ] [-q minMapQ] in.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.
-6 Assume the quality is in the Illumina 1.3+
encoding. -A Do not skip anomalous read pairs in
-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.
-b FILE List of input BAM files, one file per line [null]
-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. 
-d INT At a position, read maximally INT reads per input
-E Extended BAQ computation. This option helps
sensitivity especially for MNPs, but may hurt
specificity a little bit.
-f FILE The faidx-indexed reference file in the FASTA
format. The file can be optionally compressed by
-l FILE BED or position list file containing a list of
regions or sites where pileup or BCF should be
-q INT Minimum mapping quality for an alignment to be used
-Q INT Minimum base quality for a base to be considered
-r STR Only generate pileup in region STR [all sites]
-D Output per-sample read depth
-g Compute genotype likelihoods and output them in the
binary call format (BCF).
-S Output per-sample Phred-scaled strand bias P-value
-u Similar to -g except that the output is
uncompressed BCF, which is preferred for piping.
Options for Genotype Likelihood Computation (for -g or -u):
-e INT Phred-scaled gap extension sequencing error
probability. Reducing INT leads to longer indels.
-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. 
-I Do not perform INDEL calling
-L INT Skip INDEL calling if the average per-sample depth
is above INT. 
-o INT Phred-scaled gap open sequencing error probability.
Reducing INT leads to more indel calls. 
-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
sequencing technologies that have low indel error
rate such as ILLUMINA. [all]
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.
cat samtools 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 to reheader which enables
fast BAM concatenation.
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
-o Output the final alignment to the standard output.
-n Sort by read names rather than by chromosomal
-m INT Approximately the maximum required memory.
merge samtools 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.
-1 Use zlib compression level 1 to comrpess the output
-f Force to overwrite the output file if present.
-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
-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.
-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
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
fixmate samtools fixmate <in.nameSrt.bam> <out.bam>
Fill in mate coordinates, ISIZE and mate related flags from a
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).
-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 [-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.
-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. 
-r Compute the BQ tag (without -A) or cap base quality
by BAQ (with -A).
-E Extended BAQ calculation. This option trades
specificity for sensitivity, though the effect is
targetcut samtools 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.
phase samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q
Call and phase heterozygous SNPs. OPTIONS:
-A Drop reads with ambiguous phase.
-b STR Prefix of BAM output. When this option is in use,
phase-0 reads will be saved in file STR.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 in
-F Do not attempt to fix chimeric reads.
-k INT Maximum length for local phasing. 
-q INT Minimum Phred-scaled LOD to call a heterozygote. 
-Q INT Minimum base quality to be used in het calling. 
BCFTOOLS COMMANDS AND OPTIONS
view bcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s
listSample] [-i gapSNPratio] [-t mutRate] [-p varThres] [-P
prior] [-1 nGroup1] [-d minFrac] [-U nPerm] [-X permThres]
[-T trioType] in.bcf [region]
Convert between BCF and VCF, call variant candidates and
estimate allele frequencies.
-A Retain all possible alternate alleles at variant
sites. By default, the view command discards
-b Output in the BCF format. The default is VCF.
-D FILE Sequence dictionary (list of chromosome names) for
VCF->BCF conversion [null]
-F Indicate PL is generated by r921 or before
(ordering is different).
-G Suppress all individual genotype information.
-l FILE List of sites at which information are outputted
-N Skip sites where the REF field is not A/C/G/T
-Q Output the QCALL likelihood format
-s FILE List 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]
-S The input is VCF instead of BCF.
-u Uncompressed BCF output (force -b).
Consensus/Variant Calling Options:
-c Call variants using Bayesian inference. This option
automatically invokes option -e.
-d FLOAT When -v is in use, skip loci where the fraction of
samples covered by reads is below FLOAT. 
-e Perform max-likelihood inference only, including
estimating the site allele frequency, testing
Hardy-Weinberg equlibrium and testing associations
-g Call per-sample genotypes at variant sites (force
-i FLOAT Ratio of INDEL-to-SNP mutation rate [0.15]
-p FLOAT A site is considered to be a variant if
-P STR Prior 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 FLOAT Scaled muttion rate for variant calling [0.001]
-T STR Enable 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]
-v Output 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. 
-U INT Number of permutations for association test
(effective only with -1) 
-X FLOAT Only perform permutations for P(chi^2)<FLOAT
(effective only with -U) [0.01]
index bcftools index in.bcf
Index sorted BCF for random access.
cat bcftools cat in1.bcf [in2.bcf [...]]]
Concatenate BCF files. The input files are required to be
sorted and have identical samples appearing in the same
Sequence Alignment/Map (SAM) format 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 template/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 │ TLEN │ inferred Template LENgth (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 │
where the second column gives the string representation of the FLAG
The Variant Call Format (VCF) is a TAB-delimited format with each data
line consists of the following fields:
│Col │ Field │ Description │
│ 1 │ CHROM │ CHROMosome name │
│ 2 │ POS │ the left-most POSition of the variant │
│ 3 │ ID │ unique variant IDentifier │
│ 4 │ REF │ the REFerence allele │
│ 5 │ ALT │ the ALTernate allele(s), separated by comma │
│ 6 │ QUAL │ variant/reference QUALity │
│ 7 │ FILTER │ FILTers applied │
│ 8 │ INFO │ INFOrmation related to the variant, separated by semi-colon │
│ 9 │ FORMAT │ FORMAT of the genotype fields, separated by colon (optional) │
│10+ │ SAMPLE │ SAMPLE genotypes and per-sample information (optional) │
The following table gives the INFO tags used by samtools and bcftools.
│ Tag │ Format │ Description │
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
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 from 454.bam will be attached
o Call SNPs and short INDELs for one diploid individual:
samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - >
bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 >
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.
o Generate the consensus sequence for one diploid individual:
samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - |
vcfutils.pl vcf2fq > cns.fq
o Call somatic mutations from a pair of samples:
samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -
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.
o Call 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.
o Phase one individual:
samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - >
The calmd command is used to reduce false heterozygotes around
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 >
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
o Derive the allele frequency spectrum (AFS) on a list of sites from
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.
o Unaligned words used in bam_import.c, bam_endian.h, bam.c and
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.
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
Samtools website: <http://samtools.sourceforge.net>