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] <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
+ 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.
`samtools faidx <ref.fa>', the resultant index file
<ref.fa>.fai can be used as this <in.ref_list> 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 <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 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] <in.bam>|<in.sam>
+ pileup samtools pileup [-2sSBicv] [-f in.ref.fasta] [-t in.ref_list]
+ [-l in.site_list] [-C capMapQ] [-M maxMapQ] [-T theta] [-N
+ nHap] [-r pairDiffRate] [-m mask] [-d maxIndelDepth] [-G
+ indelPrior] <in.bam>|<in.sam>
Print the alignment in the pileup format. In the pileup for-
mat, each line represents a genomic position, consisting of
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-
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
- <in.alignment> 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
+ <in.alignment> 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 <in.header.sam> <in.bam>
[500000000]
- merge samtools merge [-h inh.sam] [-nr] <out.bam> <in1.bam>
- <in2.bam> [...]
+ merge samtools merge [-nur] [-h inh.sam] [-R reg] <out.bam>
+ <in1.bam> <in2.bam> [...]
Merge multiple sorted alignments. The header reference lists
of all the input BAM files, and the @SQ headers of inh.sam,
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 <aln.bam>
-S Treat paired-end reads and single-end reads.
- calmd samtools calmd [-eubS] <aln.bam> <ref.fasta>
+ calmd samtools calmd [-eubS
r] [-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 dif-
-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
|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.
-samtools-0.1.8 11 July 2010 samtools(1)
+samtools-0.1.9 27 October 2010 samtools(1)
projects / samtools.git / blobdiff