.TH samtools 1 "15 March 2013" "samtools-0.1.19" "Bioinformatics tools"
.SH NAME
.PP
samtools - Utilities for the Sequence Alignment/Map (SAM) format

bcftools - Utilities for the Binary Call Format (BCF) and VCF
.SH SYNOPSIS
.PP
samtools view -bt ref_list.txt -o aln.bam aln.sam.gz
.PP
samtools sort aln.bam aln.sorted
.PP
samtools index aln.sorted.bam
.PP
samtools idxstats aln.sorted.bam
.PP
samtools view aln.sorted.bam chr2:20,100,000-20,200,000
.PP
samtools merge out.bam in1.bam in2.bam in3.bam
.PP
samtools faidx ref.fasta
.PP
samtools pileup -vcf ref.fasta aln.sorted.bam
.PP
samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
.PP
samtools tview aln.sorted.bam ref.fasta
.PP
bcftools index in.bcf
.PP
bcftools view in.bcf chr2:100-200 > out.vcf
.PP
bcftools view -Nvm0.99 in.bcf > out.vcf 2> out.afs

.SH DESCRIPTION
.PP
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.

.SH SAMTOOLS COMMANDS AND OPTIONS

.TP 10
.B 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.

.B OPTIONS:
.RS
.TP 10
.B -b
Output in the BAM format.
.TP
.BI -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]
.TP
.BI -F \ INT
Skip alignments with bits present in INT [0]
.TP
.B -h
Include the header in the output.
.TP
.B -H
Output the header only.
.TP
.BI -l \ STR
Only output reads in library STR [null]
.TP
.BI -o \ FILE
Output file [stdout]
.TP
.BI -q \ INT
Skip alignments with MAPQ smaller than INT [0]
.TP
.BI -r \ STR
Only output reads in read group STR [null]
.TP
.BI -R \ FILE
Output reads in read groups listed in
.I FILE
[null]
.TP
.BI -s \ FLOAT
Fraction of templates/pairs to subsample; the integer part is treated as the
seed for the random number generator [-1]
.TP
.B -S
Input is in SAM. If @SQ header lines are absent, the
.B `-t'
option is required.
.TP
.B -c
Instead of printing the alignments, only count them and print the
total number. All filter options, such as
.B `-f',
.B `-F'
and
.B `-q'
, are taken into account.
.TP
.BI -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
.I <ref.fa>.fai
can be used as this
.I <in.ref_list>
file.
.TP
.B -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.
.RE

.TP
.B tview
samtools tview 
.RB [ \-p 
.IR chr:pos ]
.RB [ \-s 
.IR STR ]
.RB [ \-d 
.IR display ] 
.RI <in.sorted.bam> 
.RI [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.

.B Options:
.RS
.TP 14
.BI -d \ display
Output as (H)tml or (C)urses or (T)ext
.TP
.BI -p \ chr:pos
Go directly to this position
.TP
.BI -s \ STR
Display only reads from this sample or read group
.RE

.TP
.B mpileup
samtools mpileup
.RB [ \-EBugp ]
.RB [ \-C
.IR capQcoef ]
.RB [ \-r
.IR reg ]
.RB [ \-f
.IR in.fa ]
.RB [ \-l
.IR list ]
.RB [ \-M
.IR capMapQ ]
.RB [ \-Q
.IR minBaseQ ]
.RB [ \-q
.IR minMapQ ]
.I in.bam
.RI [ in2.bam
.RI [ ... ]]

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
.BR -u or -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.

.B Input Options:
.RS
.TP 10
.B -6
Assume the quality is in the Illumina 1.3+ encoding.
.B -A
Do not skip anomalous read pairs in variant calling.
.TP
.B -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.
.TP
.BI -b \ FILE
List of input BAM files, one file per line [null]
.TP
.BI -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]
.TP
.BI -d \ INT
At a position, read maximally
.I INT
reads per input BAM. [250]
.TP
.B -E
Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt
specificity a little bit.
.TP
.BI -f \ FILE
The
.BR faidx -indexed
reference file in the FASTA format. The file can be optionally compressed by
.BR razip .
[null]
.TP
.BI -l \ FILE
BED or position list file containing a list of regions or sites where pileup or BCF should be generated [null]
.TP
.BI -q \ INT
Minimum mapping quality for an alignment to be used [0]
.TP
.BI -Q \ INT
Minimum base quality for a base to be considered [13]
.TP
.BI -r \ STR
Only generate pileup in region
.I STR
[all sites]
.TP
.B Output Options:

.TP
.B -D
Output per-sample read depth
.TP
.B -g
Compute genotype likelihoods and output them in the binary call format (BCF).
.TP
.B -S
Output per-sample Phred-scaled strand bias P-value
.TP
.B -u
Similar to
.B -g
except that the output is uncompressed BCF, which is preferred for piping.

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

.TP
.BI -e \ INT
Phred-scaled gap extension sequencing error probability. Reducing
.I INT
leads to longer indels. [20]
.TP
.BI -h \ INT
Coefficient for modeling homopolymer errors. Given an
.IR l -long
homopolymer
run, the sequencing error of an indel of size
.I s
is modeled as
.IR INT * s / l .
[100]
.TP
.B -I
Do not perform INDEL calling
.TP
.BI -L \ INT
Skip INDEL calling if the average per-sample depth is above
.IR INT .
[250]
.TP
.BI -o \ INT
Phred-scaled gap open sequencing error probability. Reducing
.I INT
leads to more indel calls. [40]
.TP
.BI -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.
.TP
.BI -P \ STR
Comma dilimited list of platforms (determined by
.BR @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]
.RE

.TP
.B reheader
samtools reheader <in.header.sam> <in.bam>

Replace the header in
.I in.bam
with the header in
.I in.header.sam.
This command is much faster than replacing the header with a
BAM->SAM->BAM conversion.

.TP
.B 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
.B reheader
which enables fast BAM concatenation.

.TP
.B sort
samtools sort [-nof] [-m maxMem] <in.bam> <out.prefix>

Sort alignments by leftmost coordinates. File
.I <out.prefix>.bam
will be created. This command may also create temporary files
.I <out.prefix>.%d.bam
when the whole alignment cannot be fitted into memory (controlled by
option -m).

.B OPTIONS:
.RS
.TP 8
.B -o
Output the final alignment to the standard output.
.TP
.B -n
Sort by read names rather than by chromosomal coordinates
.TP
.B -f
Use
.I <out.prefix>
as the full output path and do not append
.I .bam
suffix.
.TP
.BI -m \ INT
Approximately the maximum required memory. [500000000]
.RE

.TP
.B 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
.IR inh.sam ,
if any, must all refer to the same set of reference sequences.
The header reference list and (unless overridden by
.BR -h )
`@' headers of
.I in1.bam
will be copied to
.IR out.bam ,
and the headers of other files will be ignored.

.B OPTIONS:
.RS
.TP 8
.B -1
Use zlib compression level 1 to comrpess the output
.TP
.B -f
Force to overwrite the output file if present.
.TP 8
.BI -h \ FILE
Use the lines of
.I FILE
as `@' headers to be copied to
.IR out.bam ,
replacing any header lines that would otherwise be copied from
.IR in1.bam .
.RI ( FILE
is actually in SAM format, though any alignment records it may contain
are ignored.)
.TP
.B -n
The input alignments are sorted by read names rather than by chromosomal
coordinates
.TP
.BI -R \ STR
Merge files in the specified region indicated by
.I STR
[null]
.TP
.B -r
Attach an RG tag to each alignment. The tag value is inferred from file names.
.TP
.B -u
Uncompressed BAM output
.RE

.TP
.B index
samtools index <aln.bam>

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

.TP
.B 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.

.TP
.B 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,
.B faidx
will index the file and create
.I <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
.B RAZF
format.

.TP
.B fixmate
samtools fixmate <in.nameSrt.bam> <out.bam>

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

.TP
.B 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
.B 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).

.B OPTIONS:
.RS
.TP 8
.B -s
Remove duplicate for single-end reads. By default, the command works for
paired-end reads only.
.TP 8
.B -S
Treat paired-end reads and single-end reads.
.RE

.TP
.B 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.

.B OPTIONS:
.RS
.TP 8
.B -A
When used jointly with
.B -r
this option overwrites the original base quality.
.TP 8
.B -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.
.TP
.B -u
Output uncompressed BAM
.TP
.B -b
Output compressed BAM
.TP
.B -S
The input is SAM with header lines
.TP
.BI -C \ INT
Coefficient to cap mapping quality of poorly mapped reads. See the
.B pileup
command for details. [0]
.TP
.B -r
Compute the BQ tag (without -A) or cap base quality by BAQ (with -A).
.TP
.B -E
Extended BAQ calculation. This option trades specificity for sensitivity, though the
effect is minor.
.RE

.TP
.B 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
.B -f
is in use, BAQ will be applied. This command is
.B only
designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].
.RE

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

Call and phase heterozygous SNPs.
.B OPTIONS:
.RS
.TP 8
.B -A
Drop reads with ambiguous phase.
.TP 8
.BI -b \ STR
Prefix of BAM output. When this option is in use, phase-0 reads will be saved in file
.BR STR .0.bam
and phase-1 reads in
.BR 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
.BR STR .chimeric.bam.
[null]
.TP
.B -F
Do not attempt to fix chimeric reads.
.TP
.BI -k \ INT
Maximum length for local phasing. [13]
.TP
.BI -q \ INT
Minimum Phred-scaled LOD to call a heterozygote. [40]
.TP
.BI -Q \ INT
Minimum base quality to be used in het calling. [13]
.RE

.SH BCFTOOLS COMMANDS AND OPTIONS

.TP 10
.B view
.B bcftools view
.RB [ \-AbFGNQSucgv ]
.RB [ \-D
.IR seqDict ]
.RB [ \-l
.IR listLoci ]
.RB [ \-s
.IR listSample ]
.RB [ \-i
.IR gapSNPratio ]
.RB [ \-t
.IR mutRate ]
.RB [ \-p
.IR varThres ]
.RB [ \-m
.IR varThres ]
.RB [ \-P
.IR prior ]
.RB [ \-1
.IR nGroup1 ]
.RB [ \-d
.IR minFrac ]
.RB [ \-U
.IR nPerm ]
.RB [ \-X
.IR permThres ]
.RB [ \-T
.IR trioType ]
.I in.bcf
.RI [ region ]

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

.RS
.TP
.B Input/Output Options:
.TP 10
.B -A
Retain all possible alternate alleles at variant sites. By default, the view
command discards unlikely alleles.
.TP 10
.B -b
Output in the BCF format. The default is VCF.
.TP
.BI -D \ FILE
Sequence dictionary (list of chromosome names) for VCF->BCF conversion [null]
.TP
.B -F
Indicate PL is generated by r921 or before (ordering is different).
.TP
.B -G
Suppress all individual genotype information.
.TP
.BI -l \ FILE
List of sites at which information are outputted [all sites]
.TP
.B -N
Skip sites where the REF field is not A/C/G/T
.TP
.B -Q
Output the QCALL likelihood format
.TP
.BI -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
.IR FILE .
[null]
.TP
.B -S
The input is VCF instead of BCF.
.TP
.B -u
Uncompressed BCF output (force -b).
.TP
.B Consensus/Variant Calling Options:
.TP 10
.B -c
Call variants using Bayesian inference. This option automatically invokes option
.BR -e .
.TP
.BI -d \ FLOAT
When
.B -v
is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0]
.TP
.B -e
Perform max-likelihood inference only, including estimating the site allele frequency,
testing Hardy-Weinberg equlibrium and testing associations with LRT.
.TP
.B -g
Call per-sample genotypes at variant sites (force -c)
.TP
.BI -i \ FLOAT
Ratio of INDEL-to-SNP mutation rate [0.15]
.TP
.BI -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]
.TP
.BI -p \ FLOAT
A site is considered to be a variant if P(ref|D)<FLOAT [0.5]
.TP
.BI -P \ STR
Prior or initial allele frequency spectrum. If STR can be
.IR full ,
.IR cond2 ,
.I flat
or the file consisting of error output from a previous variant calling
run.
.TP
.BI -t \ FLOAT
Scaled muttion rate for variant calling [0.001]
.TP
.BI -T \ STR
Enable pair/trio calling. For trio calling, option
.B -s
is usually needed to be applied to configure the trio members and their ordering.
In the file supplied to the option
.BR -s ,
the first sample must be the child, the second the father and the third the mother.
The valid values of
.I 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]
.TP
.B -v
Output variant sites only (force -c)
.TP
.B Contrast Calling and Association Test Options:
.TP
.BI -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]
.TP
.BI -U \ INT
Number of permutations for association test (effective only with
.BR -1 )
[0]
.TP
.BI -X \ FLOAT
Only perform permutations for P(chi^2)<FLOAT (effective only with
.BR -U )
[0.01]
.RE

.TP
.B index
.B bcftools index
.I in.bcf

Index sorted BCF for random access.
.RE

.TP
.B cat
.B bcftools cat
.I in1.bcf
.RI [ "in2.bcf " [ ... "]]]"

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

.TS
center box;
cb | cb | cb
n | l | l .
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
.TE

.PP
Each bit in the FLAG field is defined as:

.TS
center box;
cb | cb | cb
l | c | l .
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
.TE

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

.SH VCF FORMAT

The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:
.TS
center box;
cb | cb | cb
n | l | l .
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)
.TE

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

.TS
center box;
cb | cb | cb
l | l | l .
Tag	Format	Description
_
AF1	double	Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele
DP	int	Raw read depth (without quality filtering)
DP4	int[4]	# high-quality reference forward bases, ref reverse, alternate for and alt rev bases
FQ	int	Consensus quality. Positive: sample genotypes different; negative: otherwise
MQ	int	Root-Mean-Square mapping quality of covering reads
PC2	int[2]	Phred probability of AF in group1 samples being larger (,smaller) than in group2
PCHI2	double	Posterior weighted chi^2 P-value between group1 and group2 samples
PV4	double[4]	P-value for strand bias, baseQ bias, mapQ bias and tail distance bias
QCHI2	int	Phred-scaled PCHI2
RP	int	# permutations yielding a smaller PCHI2
CLR	int	Phred log ratio of genotype likelihoods with and without the trio/pair constraint
UGT	string	Most probable genotype configuration without the trio constraint
CGT	string	Most probable configuration with the trio constraint
VDB	float	Tests variant positions within reads. Intended for filtering RNA-seq artifacts around splice sites
RPB	float	Mann-Whitney rank-sum test for tail distance bias
HWE	float	Hardy-Weinberg equilibrium test, Wigginton et al., PMID: 15789306
.TE

.SH EXAMPLES
.IP o 2
Import SAM to BAM when
.B @SQ
lines are present in the header:

  samtools view -bS aln.sam > aln.bam

If
.B @SQ
lines are absent:

  samtools faidx ref.fa
  samtools view -bt ref.fa.fai aln.sam > aln.bam

where
.I ref.fa.fai
is generated automatically by the
.B faidx
command.

.IP o 2
Attach the
.B 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
.B RG
tag is determined by the file name the read is coming from. In this
example, in the
.IR merged.bam ,
reads from
.I ga.bam
will be attached 
.IR RG:Z:ga ,
while reads from
.I 454.bam
will be attached
.IR RG:Z:454 .

.IP o 2
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
.B -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
.B -C50
to
.B mpileup
if mapping quality is overestimated for reads containing excessive
mismatches. Applying this option usually helps
.B BWA-short
but may not other mappers.

.IP o 2
Generate the consensus sequence for one diploid individual:

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

.IP o 2
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,
.I 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
.I CLR
is effectively a score measuring the confidence of somatic calls. The higher the better.

.IP o 2
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
.I samples.txt
should consist of three lines specifying the member and order of samples (in the order of child-father-mother).
Similarly,
.I CLR
gives the Phred-log likelihood ratio with and without the trio constraint.
.I UGT
shows the most likely genotype configuration without the trio constraint, and
.I CGT
gives the most likely genotype configuration satisfying the trio constraint.

.IP o 2
Phase one individual:

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

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

.IP o 2
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
.B SM
tags in the
.B @RG
header lines. Individuals can be pooled in one alignment file; one
individual can also be separated into multiple files. The
.B -P
option specifies that indel candidates should be collected only from
read groups with the
.B @RG-PL
tag set to
.IR 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
.IR SnpGap
filter and then applying some machine learning approach

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

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

.IP o 2
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
.I sites.list
contains the list of sites with each line consisting of the reference
sequence name and position. The following
.B bcftools
commands estimate AFS by EM.

.IP o 2
Dump BAQ applied alignment for other SNP callers:

  samtools calmd -bAr aln.bam > aln.baq.bam

It adds and corrects the
.B NM
and
.B MD
tags at the same time. The
.B calmd
command also comes with the
.B -C
option, the same as the one in
.B pileup
and
.BR mpileup .
Apply if it helps.

.SH LIMITATIONS
.PP
.IP o 2
Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
.IP o 2
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.

.SH AUTHOR
.PP
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.

.SH SEE ALSO
.PP
Samtools website: <http://samtools.sourceforge.net>
.br
Samtools latest source: <https://github.com/samtools/samtools>
.br
VCFtools website with stable link to VCF specification: <http://vcftools.sourceforge.net>
.br
HTSlib website: <https://github.com/samtools/htslib>