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PROGRAM:

NAME

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

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

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 -Nvm0.99 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

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 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 FLOAT Fraction of templates/pairs to subsample; the integer part is treated
as the seed for the random number generator [-1]

-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 [-p chr:pos] [-s STR] [-d display] <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.

Options:

-d display Output as (H)tml or (C)urses or (T)ext

-p chr:pos Go directly to this position

-s STR Display only reads from this sample or read group

mpileup samtools mpileup [-EBugp] [-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:

-6 Assume the quality is in the Illumina 1.3+ encoding. -A Do not skip
anomalous read pairs in variant calling.

-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. [0]

-d INT At a position, read maximally INT reads per input BAM. [250]

-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 razip. [null]

-l FILE BED or position list file containing a list of regions or sites where
pileup or BCF should be generated [null]

-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]

Output Options:

-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. [20]

-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]

-I Do not perform INDEL calling

-L INT Skip INDEL calling if the average per-sample depth is above INT.
[250]

-o INT Phred-scaled gap open sequencing error probability. Reducing INT leads
to more indel calls. [40]

-p Apply -m and -F thresholds per sample to increase sensitivity of
calling. By default both options are applied to reads pooled from all
samples.

-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 [-nof] [-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

-f Use <out.prefix> as the full output path and do not append .bam suffix.

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

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.

OPTIONS:

-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 [null]

-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 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 [-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:

-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 -A) or cap base quality by BAQ (with -A).

-E Extended BAQ calculation. This option trades specificity for
sensitivity, though the effect is minor.

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. (2010)].

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

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 STR.chimeric.bam. [null]

-F Do not attempt to fix chimeric reads.

-k INT Maximum length for local phasing. [13]

-q INT Minimum Phred-scaled LOD to call a heterozygote. [40]

-Q INT Minimum base quality to be used in het calling. [13]

BCFTOOLS COMMANDS AND OPTIONS

view bcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s listSample] [-i
gapSNPratio] [-t mutRate] [-p varThres] [-m 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.

Input/Output Options:

-A Retain all possible alternate alleles at variant sites. By default,
the view command discards unlikely alleles.

-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 [all sites]

-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. [0]

-e Perform max-likelihood inference only, including estimating the site
allele frequency, testing Hardy-Weinberg equlibrium and testing
associations with LRT.

-g Call per-sample genotypes at variant sites (force -c)

-i FLOAT Ratio of INDEL-to-SNP mutation rate [0.15]

-m FLOAT New model for improved multiallelic and rare-variant calling. Another
ALT allele is accepted if P(chi^2) of LRT exceeds the FLOAT threshold.
The parameter seems robust and the actual value usually does not
affect the results much; a good value to use is 0.99. This is the
recommended calling method. [0]

-p FLOAT A site is considered to be a variant if P(ref|D)<FLOAT [0.5]

-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. [0]

-U INT Number of permutations for association test (effective only with -1)
[0]

-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 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:

┌────┬───────┬──────────────────────────────────────────────────────────┐
│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 field.

VCF FORMAT

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 │
├──────┼───────────┼────────────────────────────────────────────────────────────────────────────────────────────────────┤
└──────┴───────────┴────────────────────────────────────────────────────────────────────────────────────────────────────┘

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: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 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
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 - > 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.

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 - > phase.out

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

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.

Note that there is a new calling model which can be invoked by

bcftools view -m0.99 ...

which fixes some severe limitations of the default method.

For filtering, best results seem to be achieved by first applying the SnpGap filter and
then applying some machine learning approach

vcf-annotate -f SnpGap=n
vcf filter ...

Both can be found in the vcftools and htslib package (links below).

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 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.

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