samtools

samtools(1)                  Bioinformatics tools                  samtools(1)



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

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.


AUTHOR
       Heng Li from the Sanger Institute wrote the C version of samtools. Bob
       Handsaker from the Broad Institute implemented the BGZF library and Jue
       Ruan from Beijing Genomics Institute wrote the RAZF library. John
       Marshall and Petr Danecek contribute to the source code and various
       people from the 1000 Genomes Project have contributed to the SAM format
       specification.


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



samtools-0.1.19                  15 March 2013                     samtools(1)