X-Git-Url: http://woldlab.caltech.edu/gitweb/?p=samtools.git;a=blobdiff_plain;f=samtools.txt;h=09c1ccdedf9be5dd1964022519948a3932d384c8;hp=20e6c15cd707d11a7d1ed41f15b9d54732cd67ec;hb=85bb95099e58e20cc03456b7528248f7baed4db4;hpb=cb12a866906ec4ac644de0e658679261c82ab098 diff --git a/samtools.txt b/samtools.txt index 20e6c15..09c1ccd 100644 --- a/samtools.txt +++ b/samtools.txt @@ -20,60 +20,73 @@ SYNOPSIS samtools faidx ref.fasta - samtools pileup -f ref.fasta aln.sorted.bam + samtools pileup -vcf ref.fasta aln.sorted.bam - samtools mpileup -f ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam + samtools mpileup -C50 -agf ref.fasta -r chr3:1,000-2,000 in1.bam + in2.bam samtools tview aln.sorted.bam ref.fasta DESCRIPTION - Samtools is a set of utilities that manipulate alignments in the BAM + 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 + 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 + 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 + 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. COMMANDS AND OPTIONS view samtools view [-bhuHS] [-t in.refList] [-o output] [-f - reqFlag] [-F skipFlag] [-q minMapQ] [-l library] [-r read- + reqFlag] [-F skipFlag] [-q minMapQ] [-l library] [-r read- Group] [-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 + 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 + 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. - -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. + -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 Input is in SAM. If @SQ header lines are absent, the `-t' option is required. @@ -85,35 +98,24 @@ COMMANDS AND OPTIONS `samtools faidx ', the resultant index file .fai can be used as this file. - -o FILE Output file [stdout] - - -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] - - -q INT Skip alignments with MAPQ smaller than INT [0] - - -l STR Only output reads in library STR [null] - - -r STR Only output reads in read group STR [null] - - -R FILE Output reads in read groups listed in FILE [null] + -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 [ref.fasta] - Text alignment viewer (based on the ncurses library). In the - viewer, press `?' for help and press `g' to check the align- - ment start from a region in the format like - `chr10:10,000,000' or `=10,000,000' when viewing the same + Text alignment viewer (based on the ncurses library). In the + viewer, press `?' for help and press `g' to check the align- + ment start from a region in the format like + `chr10:10,000,000' or `=10,000,000' when viewing the same reference sequence. - pileup samtools pileup [-f in.ref.fasta] [-t in.ref_list] [-l - in.site_list] [-iscgS2] [-T theta] [-N nHap] [-r - pairDiffRate] | + pileup samtools pileup [-2sSBicv] [-f in.ref.fasta] [-t in.ref_list] + [-l in.site_list] [-C capMapQ] [-M maxMapQ] [-T theta] [-N + nHap] [-r pairDiffRate] [-m mask] [-d maxIndelDepth] [-G + indelPrior] | Print the alignment in the pileup format. In the pileup for- mat, each line represents a genomic position, consisting of @@ -123,20 +125,18 @@ COMMANDS AND OPTIONS 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 posi- - tion. 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 sub- - sequence on the read separated by `N/S/H' CIGAR operations. - The ASCII of the character following `^' minus 33 gives the - mapping quality. A symbol `$' marks the end of a read seg- - ment. + strand, a '>' or '<' for a reference skip, `ACGTN' for a mis- + match on the forward strand and `acgtn' for a mismatch on the + reverse strand. A pattern `\+[0-9]+[ACGTNacgtn]+' indicates + there is an insertion between this reference position and the + next reference position. The length of the insertion is given + by the integer in the pattern, followed by the inserted + sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+' repre- + sents a deletion from the reference. The deleted bases will + be presented as `*' in the following lines. Also at the read + base column, a symbol `^' marks the start of a read. The + ASCII of the character following `^' minus 33 gives the map- + ping quality. A symbol `$' marks the end of a read segment. If option -c is applied, the consensus base, Phred-scaled consensus quality, SNP quality (i.e. the Phred-scaled proba- @@ -155,68 +155,109 @@ COMMANDS AND OPTIONS OPTIONS: - -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 Disable the BAQ computation. See the mpileup com- + mand for details. - -S The input file is in SAM. + -c Call the consensus sequence using SOAPsnp consensus + model. Options -T, -N, -I and -r are only effective + when -c or -g is in use. - -i Only output pileup lines containing indels. + -C INT Coefficient for downgrading the mapping quality of + poorly mapped reads. See the mpileup command for + details. [0] + + -d INT Use the first NUM reads in the pileup for indel + calling for speed up. Zero for unlimited. [1024] -f FILE The reference sequence in the FASTA format. Index file FILE.fai will be created if absent. - -M INT Cap mapping quality at INT [60] + -g Generate genotype likelihood in the binary GLFv3 + format. This option suppresses -c, -i and -s. This + option is deprecated by the mpileup command. - -m INT Filter reads with flag containing bits in INT - [1796] - - -d INT Use the first NUM reads in the pileup for indel - calling for speed up. Zero for unlimited. [0] - - -t FILE List of reference names ane sequence lengths, in - the format described for the import command. If - this option is present, samtools assumes the input - is in SAM format; otherwise it - assumes in BAM format. + -i Only output pileup lines containing indels. - -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 -s together with -l as in the default - format we may not know the mapping quality. + -I INT Phred probability of an indel in sequencing/prep. + [40] - -c Call the consensus sequence using SOAPsnp consensus - model. Options -T, -N, -I and -r are only effective - when -c or -g is in use. + -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 - -g Generate genotype likelihood in the binary GLFv3 - format. This option suppresses -c, -i and -s. + -m INT Filter reads with flag containing bits in INT + [1796] - -T FLOAT The theta parameter (error dependency coefficient) - in the maq consensus calling model [0.85] + -M INT Cap mapping quality at INT [60] -N INT Number of haplotypes in the sample (>=2) [2] - -r FLOAT Expected fraction of differences between a pair of + -r FLOAT Expected fraction of differences between a pair of haplotypes [0.001] - -I INT Phred probability of an indel in sequencing/prep. - [40] + -s Print the mapping quality as the last column. This + option makes the output easier to parse, although + this format is not space efficient. + + -S The input file is in SAM. + + -t FILE List of reference names ane sequence lengths, in + the format described for the import command. If + this option is present, samtools assumes the input + is in SAM format; otherwise it + assumes in BAM format. -s together with -l as in + the default format we may not know the mapping + quality. + + -T FLOAT The theta parameter (error dependency coefficient) + in the maq consensus calling model [0.85] - mpileup samtools mpileup [-r reg] [-f in.fa] in.bam [in2.bam [...]] + mpileup samtools mpileup [-Bug] [-C capQcoef] [-r reg] [-f in.fa] [-l + list] [-M capMapQ] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam + [...]] - Generate pileup for multiple BAM files. Consensus calling is - not implemented. + Generate BCF or pileup for one or multiple BAM files. Align- + ment records are grouped by sample identifiers in @RG header + lines. If sample identifiers are absent, each input file is + regarded as one sample. OPTIONS: - -r STR Only generate pileup in region STR [all sites] + -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. + + -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 is + 50. [0] -f FILE The reference file [null] + -g Compute genotype likelihoods and output them in the + binary call format (BCF). + + -u Similar to -g except that the output is uncompressed + BCF, which is preferred for pipeing. + + -l FILE File containing a list of sites where pileup or BCF + is outputted [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] + reheader samtools reheader @@ -243,8 +284,8 @@ COMMANDS AND OPTIONS [500000000] - merge samtools merge [-h inh.sam] [-nr] - [...] + merge 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 inh.sam, @@ -261,12 +302,16 @@ COMMANDS AND OPTIONS SAM format, though any alignment records it may con- tain are ignored.) + -R STR Merge files in the specified region indicated by STR + -r Attach an RG tag to each alignment. The tag value is inferred from file names. -n The input alignments are sorted by read names rather than by chromosomal coordinates + -u Uncompressed BAM output + index samtools index @@ -315,7 +360,7 @@ COMMANDS AND OPTIONS -S Treat paired-end reads and single-end reads. - calmd samtools calmd [-eubS] + calmd samtools calmd [-eubSr] [-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 dif- @@ -333,6 +378,12 @@ COMMANDS AND OPTIONS -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 Perform probabilistic realignment to compute BAQ, + which will be used to cap base quality. + SAM FORMAT SAM is TAB-delimited. Apart from the header lines, which are started @@ -375,26 +426,106 @@ SAM FORMAT |0x0400 | d | the read is either a PCR or an optical duplicate | +-------+-----+--------------------------------------------------+ +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:Illu- + mina\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 com- + ing 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 pileup -vcf ref.fa aln.bam > var.raw.plp + samtools.pl varFilter -D 100 var.raw.plp > var.flt.plp + awk '($3=="*"&&$6>=50)||($3!="*"&&$6>=20)' var.flt.plp > + var.final.plp + + 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 pileup if mapping quality is overestimated + for reads containing excessive mismatches. Applying this option usu- + ally helps BWA-short but may not other mappers. It also potentially + increases reference biases. + + + o Call SNPs (not short indels) for multiple diploid individuals: + + samtools mpileup -augf ref.fa *.bam | bcftools view -bcv - > + snp.raw.bcf + bcftools view snp.raw.bcf | vcfutils.pl filter4vcf -D 2000 | bgzip + > snp.flt.vcf.gz + + 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. Similarly, one may consider to + apply -C50 to mpileup. SNP calling in this way also works for single + sample and has the advantage of enabling more powerful filtering. The + drawback is the lack of short indel calling, which may be implemented + in future. + + + o Derive the allele frequency spectrum (AFS) on a list of sites from + multiple individuals: + + samtools mpileup -gf 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 -br 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 on in pileup + and mpileup. Apply if it helps. + + LIMITATIONS - o Unaligned words used in bam_import.c, bam_endian.h, bam.c and + o Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c. - o 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 + o 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. - 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 + 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 + 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 + Ruan from Beijing Genomics Institute wrote the RAZF library. Various people in the 1000 Genomes Project contributed to the SAM format speci- fication. @@ -404,4 +535,4 @@ SEE ALSO -samtools-0.1.8 11 July 2010 samtools(1) +samtools-0.1.9 27 October 2010 samtools(1)