X-Git-Url: http://woldlab.caltech.edu/gitweb/?p=samtools.git;a=blobdiff_plain;f=samtools.1;h=ba323926d83451cf4013c511cf832d6e820a76f9;hp=45e16123948c1e72ba7193a68500e4430cc0d1c4;hb=ced7709f121a00d5049d99ee8576037994aab1d1;hpb=b27e00385f41769d03a8cca4dbd71275fc9fa906 diff --git a/samtools.1 b/samtools.1 index 45e1612..ba32392 100644 --- a/samtools.1 +++ b/samtools.1 @@ -1,4 +1,4 @@ -.TH samtools 1 "6 July 2009" "samtools-0.1.5" "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 @@ -10,13 +10,17 @@ samtools sort aln.bam aln.sorted .PP samtools index aln.sorted.bam .PP +samtools idxstats aln.sorted.bam +.PP samtools view aln.sorted.bam chr2:20,100,000-20,200,000 .PP samtools merge out.bam in1.bam in2.bam in3.bam .PP samtools faidx ref.fasta .PP -samtools pileup -f ref.fasta aln.sorted.bam +samtools pileup -vcf ref.fasta aln.sorted.bam +.PP +samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam .PP samtools tview aln.sorted.bam ref.fasta @@ -33,26 +37,231 @@ 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 -server if the BAM file name starts with `ftp://'. Samtools checks the -current working directory for the index file and will download the index -upon absence. Samtools achieves random FTP file access with the `REST' -ftp command. It does not retrieve the entire alignment file unless it is -asked to do so. +Samtools is also able to open a BAM (not SAM) file on a remote FTP or +HTTP server if the BAM file name starts with `ftp://' or `http://'. +Samtools checks the current working directory for the index file and +will download the index upon absence. Samtools does not retrieve the +entire alignment file unless it is asked to do so. .SH COMMANDS AND OPTIONS .TP 10 -.B import -samtools import +.B view +samtools view [-bchuHS] [-t in.refList] [-o output] [-f reqFlag] [-F +skipFlag] [-q minMapQ] [-l library] [-r readGroup] [-R rgFile] | [region1 [...]] + +Extract/print all or sub alignments in SAM or BAM format. If no region +is specified, all the alignments will be printed; otherwise only +alignments overlapping the specified regions will be output. An +alignment may be given multiple times if it is overlapping several +regions. A region can be presented, for example, in the following +format: `chr2' (the whole chr2), `chr2:1000000' (region starting from +1,000,000bp) or `chr2:1,000,000-2,000,000' (region between 1,000,000 and +2,000,000bp including the end points). The coordinate is 1-based. + +.B OPTIONS: +.RS +.TP 8 +.B -b +Output in the BAM format. +.TP +.BI -f \ INT +Only output alignments with all bits in INT present in the FLAG +field. INT can be in hex in the format of /^0x[0-9A-F]+/ [0] +.TP +.BI -F \ INT +Skip alignments with bits present in INT [0] +.TP +.B -h +Include the header in the output. +.TP +.B -H +Output the header only. +.TP +.BI -l \ STR +Only output reads in library STR [null] +.TP +.BI -o \ FILE +Output file [stdout] +.TP +.BI -q \ INT +Skip alignments with MAPQ smaller than INT [0] +.TP +.BI -r \ STR +Only output reads in read group STR [null] +.TP +.BI -R \ FILE +Output reads in read groups listed in +.I FILE +[null] +.TP +.B -S +Input is in SAM. If @SQ header lines are absent, the +.B `-t' +option is required. +.TP +.B -c +Instead of printing the alignments, only count them and print the +total number. All filter options, such as +.B `-f', +.B `-F' +and +.B `-q' +, are taken into account. +.TP +.BI -t \ FILE +This file is TAB-delimited. Each line must contain the reference name +and the length of the reference, one line for each distinct reference; +additional fields are ignored. This file also defines the order of the +reference sequences in sorting. If you run `samtools faidx ', +the resultant index file +.I .fai +can be used as this +.I +file. +.TP +.B -u +Output uncompressed BAM. This option saves time spent on +compression/decomprssion and is thus preferred when the output is piped +to another samtools command. +.RE + +.TP +.B tview +samtools tview [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. + +.TP +.B mpileup +samtools mpileup [-EBug] [-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. + +.B OPTIONS: +.RS +.TP 10 +.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 -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 -I +Do not perform INDEL calling +.TP +.BI -l \ FILE +File containing a list of sites where pileup or BCF is outputted [null] +.TP +.BI -L \ INT +Skip INDEL calling if the average per-sample depth is above +.IR INT . +[250] +.TP +.BI -o \ INT +Phred-scaled gap open sequencing error probability. Reducing +.I INT +leads to more indel calls. [40] +.TP +.BI -P \ 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 +.BI -q \ INT +Minimum mapping quality for an alignment to be used [0] +.TP +.BI -Q \ INT +Minimum base quality for a base to be considered [13] +.TP +.BI -r \ STR +Only generate pileup in region +.I STR +[all sites] +.TP +.B -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 +.B reheader +samtools reheader + +Replace the header in +.I in.bam +with the header in +.I in.header.sam. +This command is much faster than replacing the header with a +BAM->SAM->BAM conversion. -Since 0.1.4, this command is an alias of: +.TP +.B cat +samtools cat [-h header.sam] [-o out.bam] [ ... ] -samtools view -bt -o +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 [-n] [-m maxMem] +samtools sort [-no] [-m maxMem] Sort alignments by leftmost coordinates. File .I .bam @@ -64,29 +273,59 @@ option -m). .B OPTIONS: .RS .TP 8 +.B -o +Output the final alignment to the standard output. +.TP .B -n Sort by read names rather than by chromosomal coordinates .TP -.B -m INT +.BI -m \ INT Approximately the maximum required memory. [500000000] .RE .TP .B merge -samtools merge [-n] [...] - -Merge multiple sorted alignments. The header of -.I +samtools merge [-nur] [-h inh.sam] [-R reg] [...] + +Merge multiple sorted alignments. +The header reference lists of all the input BAM files, and the @SQ headers of +.IR inh.sam , +if any, must all refer to the same set of reference sequences. +The header reference list and (unless overridden by +.BR -h ) +`@' headers of +.I in1.bam will be copied to -.I +.IR out.bam , and the headers of other files will be ignored. .B OPTIONS: .RS .TP 8 +.BI -h \ FILE +Use the lines of +.I FILE +as `@' headers to be copied to +.IR out.bam , +replacing any header lines that would otherwise be copied from +.IR in1.bam . +.RI ( FILE +is actually in SAM format, though any alignment records it may contain +are ignored.) +.TP +.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 @@ -98,90 +337,153 @@ Index sorted alignment for fast random access. Index file will be created. .TP -.B view -samtools view [-bhuHS] [-t in.refList] [-o output] [-f reqFlag] [-F -skipFlag] [-q minMapQ] [-l library] [-r readGroup] | [region1 [...]] +.B idxstats +samtools idxstats -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', `chr2:1000000' or `chr2:1,000,000-2,000,000'. The -coordinate is 1-based. +Retrieve and print stats in the index file. The output is TAB delimited +with each line consisting of reference sequence name, sequence length, # +mapped reads and # unmapped reads. + +.TP +.B faidx +samtools faidx [region1 [...]] + +Index reference sequence in the FASTA format or extract subsequence from +indexed reference sequence. If no region is specified, +.B faidx +will index the file and create +.I .fai +on the disk. If regions are speficified, the subsequences will be +retrieved and printed to stdout in the FASTA format. The input file can +be compressed in the +.B RAZF +format. + +.TP +.B fixmate +samtools fixmate + +Fill in mate coordinates, ISIZE and mate related flags from a +name-sorted alignment. + +.TP +.B rmdup +samtools rmdup [-sS] + +Remove potential PCR duplicates: if multiple read pairs have identical +external coordinates, only retain the pair with highest mapping quality. +In the paired-end mode, this command +.B ONLY +works with FR orientation and requires ISIZE is correctly set. It does +not work for unpaired reads (e.g. two ends mapped to different +chromosomes or orphan reads). .B OPTIONS: .RS .TP 8 -.B -b -Output in the BAM format. +.B -s +Remove duplicate for single-end reads. By default, the command works for +paired-end reads only. +.TP 8 +.B -S +Treat paired-end reads and single-end reads. +.RE + .TP -.B -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. +.B calmd +samtools calmd [-EeubSr] [-C capQcoef] + +Generate the MD tag. If the MD tag is already present, this command will +give a warning if the MD tag generated is different from the existing +tag. Output SAM by default. + +.B OPTIONS: +.RS +.TP 8 +.B -A +When used jointly with +.B -r +this option overwrites the original base quality. +.TP 8 +.B -e +Convert a the read base to = if it is identical to the aligned reference +base. Indel caller does not support the = bases at the moment. .TP -.B -h -Include the header in the output. +.B -u +Output uncompressed BAM .TP -.B -H -Output the header only. +.B -b +Output compressed BAM .TP .B -S -Input is in SAM. If @SQ header lines are absent, the -.B `-t' -option is required. -.TP -.B -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 ', -the resultant index file -.I .fai -can be used as this -.I -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] +The input is SAM with header lines .TP -.B -F INT -Skip alignments with bits present in INT [0] +.BI -C \ INT +Coefficient to cap mapping quality of poorly mapped reads. See the +.B pileup +command for details. [0] .TP -.B -q INT -Skip alignments with MAPQ smaller than INT [0] +.B -r +Compute the BQ tag (without -A) or cap base quality by BAQ (with -A). .TP -.B -l STR -Only output reads in library STR [null] +.B -E +Extended BAQ calculation. This option trades specificity for sensitivity, though the +effect is minor. +.RE + .TP -.B -r STR -Only output reads in read group STR [null] +.B targetcut +samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] + +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 faidx -samtools faidx [region1 [...]] +.B phase +samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] -Index reference sequence in the FASTA format or extract subsequence from -indexed reference sequence. If no region is specified, -.B faidx -will index the file and create -.I .fai -on the disk. If regions are speficified, the subsequences will be -retrieved and printed to stdout in the FASTA format. The input file can -be compressed in the -.B RAZF -format. +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 [-f in.ref.fasta] [-t in.ref_list] [-l in.site_list] -[-iscgS2] [-T theta] [-N nHap] [-r pairDiffRate] | +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] +| Print the alignment in the pileup format. In the pileup format, each line represents a genomic position, consisting of chromosome name, @@ -190,169 +492,120 @@ 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 `$' marks the end of a read segment. +a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward +strand and `acgtn' for a mismatch on the reverse strand. A pattern +`\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this +reference position and the next reference position. The length of the +insertion is given by the integer in the pattern, followed by the +inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+' +represents a deletion from the reference. The deleted bases will be +presented as `*' in the following lines. Also at the read base column, a +symbol `^' marks the start of a read. The ASCII of the character +following `^' minus 33 gives the mapping quality. A symbol `$' marks the +end of a read segment. If option .B -c -is applied, the consensus base, consensus quality, SNP quality and 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. +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 -s -Print the mapping quality as the last column. This option makes the -output easier to parse, although this format is not space efficient. - +.B -B +Disable the BAQ computation. See the +.B mpileup +command for details. .TP -.B -S -The input file is in SAM. - +.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 -.B -i -Only output pileup lines containing indels. - +.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 -.B -f FILE +.BI -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] - +.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 -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 -is in SAM format; otherwise it assumes in BAM format. - +.B -i +Only output pileup lines containing indels. .TP -.B -l FILE +.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 -.B -s -together with -.B -l -as in the default format we may not know the mapping quality. - -.TP -.B -c -Call the consensus sequence using MAQ consensus model. Options -.B -T, -.B -N, -.B -I -and -.B -r -are only effective when -.B -c -or -.B -g -is in use. - .TP -.B -g -Generate genotype likelihood in the binary GLFv3 format. This option -suppresses -c, -i and -s. - +.BI -m \ INT +Filter reads with flag containing bits in +.I INT +[1796] .TP -.B -T FLOAT -The theta parameter (error dependency coefficient) in the maq consensus -calling model [0.85] - +.BI -M \ INT +Cap mapping quality at INT [60] .TP -.B -N INT +.BI -N \ INT Number of haplotypes in the sample (>=2) [2] - .TP -.B -r FLOAT +.BI -r \ FLOAT Expected fraction of differences between a pair of haplotypes [0.001] - -.TP -.B -I INT -Phred probability of an indel in sequencing/prep. [40] - -.RE - -.TP -.B tview -samtools tview [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'. Note that if the region -showed on the screen contains no mapped reads, a blank screen will be -seen. This is a known issue and will be improved later. - -.RE - .TP -.B fixmate -samtools fixmate - -Fill in mate coordinates, ISIZE and mate related flags from a -name-sorted alignment. - +.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 rmdup -samtools rmdup - -Remove potential PCR duplicates: if multiple read pairs have identical -external coordinates, only retain the pair with highest mapping quality. -This command -.B ONLY -works with FR orientation and requires ISIZE is correctly set. - -.RE - +.B -S +The input file is in SAM. .TP -.B rmdupse -samtools rmdupse - -Remove potential duplicates for single-ended reads. This command will -treat all reads as single-ended even if they are paired in fact. - -.RE - +.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 +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 -.B fillmd -samtools fillmd [-e] - -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. - -.B OPTIONS: -.RS -.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. - +.BI -T \ FLOAT +The theta parameter (error dependency coefficient) in the maq consensus +calling model [0.85] .RE .SH SAM FORMAT @@ -385,38 +638,180 @@ Each bit in the FLAG field is defined as: .TS center box; -cb | cb -l | l . -Flag Description +cb | cb | cb +l | c | l . +Flag Chr Description _ -0x0001 the read is paired in sequencing -0x0002 the read is mapped in a proper pair -0x0004 the query sequence itself is unmapped -0x0008 the mate is unmapped -0x0010 strand of the query (1 for reverse) -0x0020 strand of the mate -0x0040 the read is the first read in a pair -0x0080 the read is the second read in a pair -0x0100 the alignment is not primary -0x0200 the read fails platform/vendor quality checks -0x0400 the read is either a PCR or an optical duplicate +0x0001 p the read is paired in sequencing +0x0002 P the read is mapped in a proper pair +0x0004 u the query sequence itself is unmapped +0x0008 U the mate is unmapped +0x0010 r strand of the query (1 for reverse) +0x0020 R strand of the mate +0x0040 1 the read is the first read in a pair +0x0080 2 the read is the second read in a pair +0x0100 s the alignment is not primary +0x0200 f the read fails platform/vendor quality checks +0x0400 d the read is either a PCR or an optical duplicate .TE +.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 Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c. .IP o 2 -CIGAR operation P is not properly handled at the moment. +In merging, the input files are required to have the same number of +reference sequences. The requirement can be relaxed. In addition, +merging does not reconstruct the header dictionaries +automatically. Endusers have to provide the correct header. Picard is +better at merging. +.IP o 2 +Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan +reads or ends mapped to different chromosomes). If this is a concern, +please use Picard's MarkDuplicate which correctly handles these cases, +although a little slower. .SH AUTHOR .PP Heng Li from the Sanger Institute wrote the C version of samtools. Bob Handsaker from the Broad Institute implemented the BGZF library and Jue -Ruan from Beijing Genomics Institute wrote the RAZF library. Various -people in the 1000Genomes 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 .PP -Samtools website: http://samtools.sourceforge.net +Samtools website: