-.TH samtools 1 "11 July 2010" "samtools-0.1.8" "Bioinformatics tools"
+.TH samtools 1 "16 March 2011" "samtools-0.1.14" "Bioinformatics tools"
.SH NAME
.PP
samtools - Utilities for the Sequence Alignment/Map (SAM) format
.PP
samtools faidx ref.fasta
.PP
-samtools pileup -f ref.fasta aln.sorted.bam
+samtools pileup -vcf ref.fasta aln.sorted.bam
.PP
-samtools mpileup -f ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
+samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
.PP
samtools tview aln.sorted.bam ref.fasta
.TP 10
.B view
-samtools view [-bhuHS] [-t in.refList] [-o output] [-f reqFlag] [-F
+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
.B -b
Output in the BAM format.
.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.
+.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.
.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
.B -S
Input is in SAM. If @SQ header lines are absent, the
.B `-t'
option is required.
.TP
-.B -t FILE
+.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
.I <in.ref_list>
file.
.TP
-.B -o FILE
-Output file [stdout]
-.TP
-.B -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
-.B -F INT
-Skip alignments with bits present in INT [0]
-.TP
-.B -q INT
-Skip alignments with MAPQ smaller than INT [0]
-.TP
-.B -l STR
-Only output reads in library STR [null]
-.TP
-.B -r STR
-Only output reads in read group STR [null]
-.TP
-.B -R FILE
-Output reads in read groups listed in
-.I FILE
-[null]
+.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
viewing the same reference sequence.
.TP
-.B pileup
-samtools pileup [-f in.ref.fasta] [-t in.ref_list] [-l in.site_list]
-[-iscgS2] [-T theta] [-N nHap] [-r pairDiffRate] <in.bam>|<in.sam>
-
-Print the alignment in the pileup format. In the pileup format, 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,
-`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 segment
-which is a contiguous subsequence on the read separated by `N/S/H' CIGAR
-operations. The ASCII of the character following `^' minus 33 gives the
-mapping quality. A symbol `$' marks the end of a read segment.
-
-If option
-.B -c
-is applied, the consensus base, Phred-scaled consensus quality, SNP
-quality (i.e. the Phred-scaled probability of the consensus being
-identical to the reference) and root mean square (RMS) mapping quality
-of the reads covering the site will be inserted between the `reference
-base' and the `read bases' columns. An indel occupies an additional
-line. Each indel line consists of chromosome name, coordinate, a star,
-the genotype, consensus quality, SNP quality, RMS mapping quality, #
-covering reads, the first alllele, the second allele, # reads supporting
-the first allele, # reads supporting the second allele and # reads
-containing indels different from the top two alleles.
+.B mpileup
+samtools mpileup [-EBug] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-M capMapQ] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]
-The position of indels is offset by -1.
+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.
.B OPTIONS:
.RS
.TP 10
-.B -s
-Print the mapping quality as the last column. This option makes the
-output easier to parse, although this format is not space efficient.
-.TP
-.B -S
-The input file is in SAM.
-.TP
-.B -i
-Only output pileup lines containing indels.
-.TP
-.B -f FILE
-The reference sequence in the FASTA format. Index file
-.I FILE.fai
-will be created if
-absent.
-.TP
-.B -M INT
-Cap mapping quality at INT [60]
-.TP
-.B -m INT
-Filter reads with flag containing bits in
-.I
-INT
-[1796]
-.TP
-.B -d INT
-Use the first
-.I NUM
-reads in the pileup for indel calling for speed up. Zero for unlimited. [0]
-.TP
-.B -t FILE
-List of reference names ane sequence lengths, in the format described
-for the
-.B import
-command. If this option is present, samtools assumes the input
-.I <in.alignment>
-is in SAM format; otherwise it assumes in BAM format.
+.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 -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 -D
+Output per-sample read depth
+.TP
+.BI -e \ INT
+Phred-scaled gap extension sequencing error probability. Reducing
+.I INT
+leads to longer indels. [20]
+.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 reference file [null]
.TP
-.B -l FILE
-List of sites at which pileup is output. This file is space
-delimited. The first two columns are required to be chromosome and
-1-based coordinate. Additional columns are ignored. It is
-recommended to use option
-.B -s
-together with
-.B -l
-as in the default format we may not know the mapping quality.
+.B -g
+Compute genotype likelihoods and output them in the binary call format (BCF).
+.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 -c
-Call the consensus sequence using SOAPsnp consensus model. Options
-.B -T,
-.B -N,
.B -I
-and
-.B -r
-are only effective when
-.B -c
-or
-.B -g
-is in use.
+Do not perform INDEL calling
.TP
-.B -g
-Generate genotype likelihood in the binary GLFv3 format. This option
-suppresses -c, -i and -s.
+.BI -l \ FILE
+File containing a list of sites where pileup or BCF is outputted [null]
.TP
-.B -T FLOAT
-The theta parameter (error dependency coefficient) in the maq consensus
-calling model [0.85]
+.BI -L \ INT
+Skip INDEL calling if the average per-sample depth is above
+.IR INT .
+[250]
.TP
-.B -N INT
-Number of haplotypes in the sample (>=2) [2]
+.BI -o \ INT
+Phred-scaled gap open sequencing error probability. Reducing
+.I INT
+leads to more indel calls. [40]
.TP
-.B -r FLOAT
-Expected fraction of differences between a pair of haplotypes [0.001]
+.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]
.TP
-.B -I INT
-Phred probability of an indel in sequencing/prep. [40]
-.RE
-
+.BI -q \ INT
+Minimum mapping quality for an alignment to be used [0]
.TP
-.B mpileup
-samtools mpileup [-r reg] [-f in.fa] in.bam [in2.bam [...]]
-
-Generate pileup for multiple BAM files. Consensus calling is not
-implemented.
-
-.B OPTIONS:
-.RS
-.TP 8
-.B -r STR
+.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 -f FILE
-The reference file [null]
+.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.
.RE
.TP
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 [-no] [-m maxMem] <in.bam> <out.prefix>
.B -n
Sort by read names rather than by chromosomal coordinates
.TP
-.B -m INT
+.BI -m \ INT
Approximately the maximum required memory. [500000000]
.RE
.TP
.B merge
-samtools merge [-h inh.sam] [-nr] <out.bam> <in1.bam> <in2.bam> [...]
+samtools merge [-nur] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]
Merge multiple sorted alignments.
The header reference lists of all the input BAM files, and the @SQ headers of
.B OPTIONS:
.RS
.TP 8
-.B -h FILE
+.BI -h \ FILE
Use the lines of
.I FILE
as `@' headers to be copied to
is actually in SAM format, though any alignment records it may contain
are ignored.)
.TP
+.BI -R \ STR
+Merge files in the specified region indicated by
+.I STR
+.TP
.B -r
Attach an RG tag to each alignment. The tag value is inferred from file names.
.TP
.B -n
The input alignments are sorted by read names rather than by chromosomal
coordinates
+.TP
+.B -u
+Uncompressed BAM output
.RE
.TP
.TP
.B calmd
-samtools calmd [-
eubS] <aln.bam> <ref.fasta>
+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
.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 -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
+
+.TP
+.B pileup
+samtools pileup [-2sSBicv] [-f in.ref.fasta] [-t in.ref_list] [-l
+in.site_list] [-C capMapQ] [-M maxMapQ] [-T theta] [-N nHap] [-r
+pairDiffRate] [-m mask] [-d maxIndelDepth] [-G indelPrior]
+<in.bam>|<in.sam>
+
+Print the alignment in the pileup format. In the pileup format, 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.
+
+If option
+.B -c
+is applied, the consensus base, Phred-scaled consensus quality, SNP
+quality (i.e. the Phred-scaled probability of the consensus being
+identical to the reference) and root mean square (RMS) mapping quality
+of the reads covering the site will be inserted between the `reference
+base' and the `read bases' columns. An indel occupies an additional
+line. Each indel line consists of chromosome name, coordinate, a star,
+the genotype, consensus quality, SNP quality, RMS mapping quality, #
+covering reads, the first alllele, the second allele, # reads supporting
+the first allele, # reads supporting the second allele and # reads
+containing indels different from the top two alleles.
+
+.B NOTE:
+Since 0.1.10, the `pileup' command is deprecated by `mpileup'.
+
+.B OPTIONS:
+.RS
+.TP 10
+.B -B
+Disable the BAQ computation. See the
+.B mpileup
+command for details.
+.TP
+.B -c
+Call the consensus sequence. Options
+.BR -T ", " -N ", " -I " and " -r
+are only effective when
+.BR -c " or " -g
+is in use.
+.TP
+.BI -C \ INT
+Coefficient for downgrading the mapping quality of poorly mapped
+reads. See the
+.B mpileup
+command for details. [0]
+.TP
+.BI -d \ INT
+Use the first
+.I NUM
+reads in the pileup for indel calling for speed up. Zero for unlimited. [1024]
+.TP
+.BI -f \ FILE
+The reference sequence in the FASTA format. Index file
+.I FILE.fai
+will be created if
+absent.
+.TP
+.B -g
+Generate genotype likelihood in the binary GLFv3 format. This option
+suppresses -c, -i and -s. This option is deprecated by the
+.B mpileup
+command.
+.TP
+.B -i
+Only output pileup lines containing indels.
+.TP
+.BI -I \ INT
+Phred probability of an indel in sequencing/prep. [40]
+.TP
+.BI -l \ FILE
+List of sites at which pileup is output. This file is space
+delimited. The first two columns are required to be chromosome and
+1-based coordinate. Additional columns are ignored. It is
+recommended to use option
+.TP
+.BI -m \ INT
+Filter reads with flag containing bits in
+.I INT
+[1796]
+.TP
+.BI -M \ INT
+Cap mapping quality at INT [60]
+.TP
+.BI -N \ INT
+Number of haplotypes in the sample (>=2) [2]
+.TP
+.BI -r \ FLOAT
+Expected fraction of differences between a pair of haplotypes [0.001]
+.TP
+.B -s
+Print the mapping quality as the last column. This option makes the
+output easier to parse, although this format is not space efficient.
+.TP
+.B -S
+The input file is in SAM.
+.TP
+.BI -t \ FILE
+List of reference names ane sequence lengths, in the format described
+for the
+.B import
+command. If this option is present, samtools assumes the input
+.I <in.alignment>
+is in SAM format; otherwise it assumes in BAM format.
+.B -s
+together with
+.B -l
+as in the default format we may not know the mapping quality.
+.TP
+.BI -T \ FLOAT
+The theta parameter (error dependency coefficient) in the maq consensus
+calling model [0.85]
.RE
.SH SAM FORMAT
0x0400 d the read is either a PCR or an optical duplicate
.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
+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.
+
+.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
.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. Various
-people in the 1000 Genomes Project contributed to the SAM format
+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
projects / samtools.git / blobdiff