Provided by: samtools_0.1.19-1ubuntu1_amd64 bug


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

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


       samtools view -bt ref_list.txt -o aln.bam aln.sam.gz

       samtools sort aln.bam aln.sorted

       samtools index aln.sorted.bam

       samtools idxstats aln.sorted.bam

       samtools view aln.sorted.bam chr2:20,100,000-20,200,000

       samtools merge out.bam in1.bam in2.bam in3.bam

       samtools faidx ref.fasta

       samtools pileup -vcf ref.fasta aln.sorted.bam

       samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam

       samtools tview aln.sorted.bam ref.fasta

       bcftools index in.bcf

       bcftools view in.bcf chr2:100-200 > out.vcf

       bcftools view -Nvm0.99 in.bcf > out.vcf 2> out.afs


       Samtools  is  a  set of utilities that manipulate alignments in the BAM format. It imports
       from and exports to the SAM (Sequence Alignment/Map) format,  does  sorting,  merging  and
       indexing, and allows to retrieve reads in any regions swiftly.

       Samtools  is  designed  to  work on a stream. It regards an input file `-' as the standard
       input (stdin) and an output file `-' as the standard output (stdout). Several commands can
       thus be combined with Unix pipes. Samtools always output warning and error messages to the
       standard error 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.


       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        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

                 -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


                 -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

                 -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.

                 -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

                 -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).


                 -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.


                 -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

                 -n      The input alignments are sorted by read names rather than by chromosomal

                 -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

                 -u      Uncompressed BAM output

       index     samtools index <aln.bam>

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

       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

       rmdup     samtools rmdup [-sS] <> <out.bam>

                 Remove potential PCR duplicates: if multiple read pairs have identical  external
                 coordinates,  only retain the pair with highest mapping quality.  In the paired-
                 end mode, this command ONLY works with FR  orientation  and  requires  ISIZE  is
                 correctly  set.  It  does  not  work for unpaired reads (e.g. two ends mapped to
                 different chromosomes or orphan reads).


                 -s      Remove duplicate for single-end reads. By default, the command works for
                         paired-end reads only.

                 -S      Treat paired-end reads and single-end reads.

       calmd     samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta>

                 Generate  the MD tag. If the MD tag is already present, this command will give a
                 warning if the MD tag generated is different from the existing tag.  Output  SAM
                 by default.


                 -A      When  used  jointly  with  -r  this  option overwrites the original base

                 -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]


       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

                 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

                 -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.

                 -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)

                 -X FLOAT  Only  perform permutations for P(chi^2)<FLOAT (effective only with -U)

       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.


       Sequence  Alignment/Map  (SAM) format is TAB-delimited. Apart from the header lines, which
       are started with the `@' symbol, each alignment line consists of:

                │ColFieldDescription                        │
                │ 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:

                   │ FlagChrDescription                    │
                   │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.


       The Variant Call Format (VCF) is a TAB-delimited format with each data  line  consists  of
       the following fields:

             │ColFieldDescription                          │
             │ 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.

│ TagFormatDescription                                             │


       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 | 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 - | vcf2fq >

       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 - >

         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 | 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.


       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.


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


       Samtools website: <>
       Samtools latest source: <>
       VCFtools website with stable link to VCF specification: <>
       HTSlib website: <>