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 -f ref.fasta aln.sorted.bam
+ 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
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
- import samtools import <in.ref_list> <in.sam> <out.bam>
+ view samtools view [-bchuHS] [-t in.refList] [-o output] [-f
+ 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
+ it is overlapping several regions. A region can be presented,
+ for example, in the following format: `chr2' (the whole
+ chr2), `chr2:1000000' (region starting from 1,000,000bp) or
+ `chr2:1,000,000-2,000,000' (region between 1,000,000 and
+ 2,000,000bp including the end points). The coordinate is
+ 1-based.
+
+ OPTIONS:
+
+ -b Output in the BAM format.
+
+ -f INT Only output alignments with all bits in INT present
+ in the FLAG field. INT can be in hex in the format of
+ /^0x[0-9A-F]+/ [0]
+
+ -F INT Skip alignments with bits present in INT [0]
+
+ -h Include the header in the output.
+
+ -H Output the header only.
+
+ -l STR Only output reads in library STR [null]
+
+ -o FILE Output file [stdout]
+
+ -q INT Skip alignments with MAPQ smaller than INT [0]
+
+ -r STR Only output reads in read group STR [null]
+
+ -R FILE Output reads in read groups listed in FILE [null]
+
+ -S Input is in SAM. If @SQ header lines are absent, the
+ `-t' option is required.
+
+ -c Instead of printing the alignments, only count them
+ and print the total number. All filter options, such
+ as `-f', `-F' and `-q' , are taken into account.
+
+ -t FILE This file is TAB-delimited. Each line must contain
+ the reference name and the length of the reference,
+ one line for each distinct reference; additional
+ fields are ignored. This file also defines the order
+ of the reference sequences in sorting. If you run
+ `samtools faidx <ref.fa>', the resultant index file
+ <ref.fa>.fai can be used as this <in.ref_list> file.
+
+ -u Output uncompressed BAM. This option saves time spent
+ on compression/decomprssion and is thus preferred
+ when the output is piped to another samtools command.
+
+
+ tview samtools tview <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
+ reference sequence.
+
+
+ mpileup samtools mpileup [-Bug] [-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. 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.
- Since 0.1.4, this command is an alias of:
+ OPTIONS:
+
+ -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 for
+ BWA is 50. [0]
+
+ -e INT Phred-scaled gap extension sequencing error probabil-
+ ity. Reducing INT leads to longer indels. [20]
+
+ -f FILE The reference file [null]
+
+ -g Compute genotype likelihoods and output them in the
+ binary call format (BCF).
+
+ -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]
+
+ -l FILE File containing a list of sites where pileup or BCF
+ is outputted [null]
+
+ -o INT Phred-scaled gap open sequencing error probability.
+ Reducing INT leads to more indel calls. [40]
+
+ -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 sequenc-
+ ing technologies that have low indel error rate such
+ as ILLUMINA. [all]
+
+ -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]
+
+ -u Similar to -g except that the output is uncompressed
+ BCF, which is preferred for piping.
- samtools view -bt <in.ref_list> -o <out.bam> <in.sam>
+ reheader samtools reheader <in.header.sam> <in.bam>
- sort samtools sort [-n] [-m maxMem] <in.bam> <out.prefix>
+ 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.
+
+
+ sort samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>
Sort alignments by leftmost coordinates. File <out.pre-
fix>.bam will be created. This command may also create tempo-
- rary files <out.prefix>.%d.bam when the whole alignment can-
+ rary files <out.prefix>.%d.bam when the whole alignment can-
not be fitted into memory (controlled by option -m).
OPTIONS:
+ -o Output the final alignment to the standard output.
+
-n Sort by read names rather than by chromosomal coordi-
nates
- -m INT Approximately the maximum required memory.
+ -m INT Approximately the maximum required memory.
[500000000]
- merge samtools merge [-h inh.sam] [-n] <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,
+ 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
+ sequences. The header reference list and (unless overridden
+ by -h) `@' headers of in1.bam will be copied to out.bam, and
the headers of other files will be ignored.
OPTIONS:
- -h FILE Use the lines of FILE as `@' headers to be copied to
+ -h FILE Use the lines of FILE as `@' headers to be copied to
out.bam, replacing any header lines that would other-
- wise be copied from in1.bam. (FILE is actually in
- SAM format, though any alignment records it may con-
+ wise be copied from in1.bam. (FILE is actually in
+ 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>
<aln.bam>.bai will be created.
- view samtools view [-bhuHS] [-t in.refList] [-o output] [-f
- reqFlag] [-F skipFlag] [-q minMapQ] [-l library] [-r read-
- Group] <in.bam>|<in.sam> [region1 [...]]
+ idxstats samtools idxstats <aln.bam>
- 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.
+ 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.
- OPTIONS:
- -b Output in the BAM format.
+ faidx samtools faidx <ref.fasta> [region1 [...]]
- -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.
+ Index reference sequence in the FASTA format or extract sub-
+ sequence 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.
- -h Include the header in the output.
- -H Output the header only.
+ fixmate samtools fixmate <in.nameSrt.bam> <out.bam>
- -S Input is in SAM. If @SQ header lines are absent, the
- `-t' option is required.
+ Fill in mate coordinates, ISIZE and mate related flags from a
+ name-sorted alignment.
- -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.
- -o FILE Output file [stdout]
+ rmdup samtools rmdup [-sS] <input.srt.bam> <out.bam>
- -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]
+ 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 com-
+ mand 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).
- -F INT Skip alignments with bits present in INT [0]
+ OPTIONS:
- -q INT Skip alignments with MAPQ smaller than INT [0]
+ -s Remove duplicate for single-end reads. By default,
+ the command works for paired-end reads only.
- -l STR Only output reads in library STR [null]
+ -S Treat paired-end reads and single-end reads.
- -r STR Only output reads in read group STR [null]
+ calmd samtools calmd [-eubSr] [-C capQcoef] <aln.bam> <ref.fasta>
- faidx samtools faidx <ref.fasta> [region1 [...]]
+ Generate the MD tag. If the MD tag is already present, this
+ command will give a warning if the MD tag generated is dif-
+ ferent from the existing tag. Output SAM by default.
- Index reference sequence in the FASTA format or extract sub-
- sequence 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.
+ OPTIONS:
+
+ -A When used jointly with -r this option overwrites the
+ original base quality.
+
+ -e Convert a the read base to = if it is identical to
+ the aligned reference base. Indel caller does not
+ support the = bases at the moment.
+
+ -u Output uncompressed BAM
+
+ -b Output compressed BAM
+
+ -S The input is SAM with header lines
+ -C INT Coefficient to cap mapping quality of poorly mapped
+ reads. See the pileup command for details. [0]
- 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>
+ -r Compute the BQ tag without changing the base quality.
- Print the alignment in the pileup format. In the pileup for-
- mat, each line represents a genomic position, consisting of
+
+ 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
chromosome name, coordinate, reference base, read bases, read
- qualities and alignment mapping qualities. Information on
+ 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, `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 `$' marks the end of a read seg-
- ment.
-
- If option -c is applied, the consensus base, Phred-scaled
- consensus quality, SNP quality (i.e. the Phred-scaled proba-
+ end of a read are all encoded at the read base column. At
+ this column, a dot stands for a match to the reference base
+ on the forward strand, a comma for a match on the reverse
+ strand, a '>' or '<' for a reference skip, `ACGTN' for a mis-
+ match on the forward strand and `acgtn' for a mismatch on the
+ reverse strand. A pattern `\+[0-9]+[ACGTNacgtn]+' indicates
+ there is an insertion between this reference position and the
+ next reference position. The length of the insertion is given
+ by the integer in the pattern, followed by the inserted
+ sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+' repre-
+ sents a deletion from the reference. The deleted bases will
+ be presented as `*' in the following lines. Also at the read
+ base column, a symbol `^' marks the start of a read. The
+ ASCII of the character following `^' minus 33 gives the map-
+ ping quality. A symbol `$' 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-
bility 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, coordi-
- nate, a star, the genotype, consensus quality, SNP quality,
+ 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, coordi-
+ nate, 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
+ second allele, # reads supporting the first allele, # reads
+ supporting the second allele and # reads containing indels
different from the top two alleles.
- 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.
+ NOTE: Since 0.1.10, the `pileup' command is deprecated by
+ `mpileup'.
+ OPTIONS:
- -S The input file is in SAM.
+ -B Disable the BAQ computation. See the mpileup com-
+ mand for details.
+ -c Call the consensus sequence. 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
+ -f FILE The reference sequence in the FASTA format. Index
file FILE.fai will be created if absent.
+ -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 Cap mapping quality at INT [60]
-
-
- -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.
+ -I INT Phred probability of an indel in sequencing/prep.
+ [40]
-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.
+ to use option
+ -m INT Filter reads with flag containing bits in INT
+ [1796]
- -c Call the consensus sequence using MAQ consensus
- model. Options -T, -N, -I and -r are only effective
- when -c or -g is in use.
+ -M INT Cap mapping quality at INT [60]
+ -N INT Number of haplotypes in the sample (>=2) [2]
- -g Generate genotype likelihood in the binary GLFv3
- format. This option suppresses -c, -i and -s.
+ -r FLOAT Expected fraction of differences between a pair of
+ haplotypes [0.001]
+ -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]
- -N INT Number of haplotypes in the sample (>=2) [2]
+SAM FORMAT
+ SAM is TAB-delimited. Apart from the header lines, which are started
+ with the `@' symbol, each alignment line consists of:
- -r FLOAT Expected fraction of differences between a pair of
- haplotypes [0.001]
+ +----+-------+----------------------------------------------------------+
+ |Col | Field | Description |
+ +----+-------+----------------------------------------------------------+
+ | 1 | QNAME | Query (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 | ISIZE | Inferred 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:
- -I INT Phred probability of an indel in sequencing/prep.
- [40]
+ +-------+-----+--------------------------------------------------+
+ | Flag | Chr | Description |
+ +-------+-----+--------------------------------------------------+
+ |0x0001 | p | the read is paired in sequencing |
+ |0x0002 | P | the read is mapped in a proper pair |
+ |0x0004 | u | the query sequence itself is unmapped |
+ |0x0008 | U | the mate is unmapped |
+ |0x0010 | r | strand of the query (1 for reverse) |
+ |0x0020 | R | strand of the mate |
+ |0x0040 | 1 | the read is the first read in a pair |
+ |0x0080 | 2 | the read is the second read in a pair |
+ |0x0100 | s | the alignment is not primary |
+ |0x0200 | f | the read fails platform/vendor quality checks |
+ |0x0400 | d | the read is either a PCR or an optical duplicate |
+ +-------+-----+--------------------------------------------------+
+EXAMPLES
+ o Import SAM to BAM when @SQ lines are present in the header:
- tview samtools tview <in.sorted.bam> [ref.fasta]
+ samtools view -bS aln.sam > aln.bam
- 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'.
+ If @SQ lines are absent:
+ samtools faidx ref.fa
+ samtools view -bt ref.fa.fai aln.sam > aln.bam
- fixmate samtools fixmate <in.nameSrt.bam> <out.bam>
+ where ref.fa.fai is generated automatically by the faidx command.
- Fill in mate coordinates, ISIZE and mate related flags from a
- name-sorted alignment.
+ o Attach the RG tag while merging sorted alignments:
- rmdup samtools rmdup <input.srt.bam> <out.bam>
+ 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
- Remove potential PCR duplicates: if multiple read pairs have
- identical external coordinates, only retain the pair with
- highest mapping quality. This command ONLY works with FR
- orientation and requires ISIZE is correctly set.
+ 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.
- rmdupse samtools rmdupse <input.srt.bam> <out.bam>
+ o Call SNPs and short indels for one diploid individual:
- Remove potential duplicates for single-ended reads. This com-
- mand will treat all reads as single-ended even if they are
- paired in fact.
+ samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - >
+ var.raw.bcf
+ bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 >
+ var.flt.vcf
+ The -D option of varFilter controls the maximum read depth, which
+ should be adjusted to about twice the average read depth. One may
+ consider to add -C50 to mpileup if mapping quality is overestimated
+ for reads containing excessive mismatches. Applying this option usu-
+ ally helps BWA-short but may not other mappers.
- fillmd samtools fillmd [-e] <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-
- ferent from the existing tag.
+ o Call SNPs and short indels for multiple diploid individuals:
- OPTIONS:
+ 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
- -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.
+ 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.
+ o Derive the allele frequency spectrum (AFS) on a list of sites from
+ multiple individuals:
-SAM FORMAT
- SAM is TAB-delimited. Apart from the header lines, which are started
- with the `@' symbol, each alignment line consists of:
+ 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.
- +----+-------+----------------------------------------------------------+
- |Col | Field | Description |
- +----+-------+----------------------------------------------------------+
- | 1 | QNAME | Query (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 | ISIZE | Inferred 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:
+ 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.
- +-------+--------------------------------------------------+
- | Flag | 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 |
- +-------+--------------------------------------------------+
LIMITATIONS
o Unaligned words used in bam_import.c, bam_endian.h, bam.c and
bam_aux.c.
- o CIGAR operation P is not properly handled at the moment.
-
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' rmdup does not work for single-end data and does not remove
- duplicates across chromosomes. Picard is better.
+ o Samtools paired-end rmdup does not work for unpaired reads (e.g.
+ orphan reads or ends mapped to different chromosomes). If this is a
+ concern, please use Picard's MarkDuplicate which correctly handles
+ these cases, although a little slower.
AUTHOR
Heng Li from the Sanger Institute wrote the C version of samtools. Bob
Handsaker from the Broad Institute implemented the BGZF library and Jue
- Ruan from Beijing Genomics Institute wrote the RAZF library. Various
- people in the 1000Genomes Project contributed to the SAM format speci-
+ Ruan from Beijing Genomics Institute wrote the RAZF library. John Mar-
+ shall and Petr Danecek contribute to the source code and various people
+ from the 1000 Genomes Project have contributed to the SAM format speci-
fication.
-samtools-0.1.7 10 November 2009 samtools(1)
+samtools-0.1.10 15 November 2010 samtools(1)
projects / samtools.git / blobdiff