samtools manual(1)
2013-12-23 17:17
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Manual Reference Pages - samtools (1)
NAME
samtools - Utilities for the Sequence Alignment/Map (SAM) formatbcftools - Utilities for the Binary Call Format (BCF) and VCF
CONTENTS
SynopsisDescription
Samtools Commands And Options
Bcftools Commands And Options
Sam Format
Vcf Format
Examples
Limitations
Author
See Also
SYNOPSIS
samtools view -bt ref_list.txt -o aln.bam aln.sam.gzsamtools sort aln.bam aln.sorted
samtools index aln.sorted.bam
samtools idxstats aln.sorted.bam
samtools view aln.sorted.bam chr2:20,100,000-20,200,000
samtools merge out.bam in1.bam in2.bam in3.bam
samtools faidx ref.fasta
samtools pileup -vcf ref.fasta aln.sorted.bam
samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
samtools tview aln.sorted.bam ref.fasta
bcftools index in.bcf
bcftools view in.bcf chr2:100-200 > out.vcf
bcftools view -vc in.bcf > out.vcf 2> out.afs
DESCRIPTION
Samtools is a set of utilities that manipulate alignments in the BAM format. It imports from and exports to the SAM (Sequence Alignment/Map) format, does sorting, merging and indexing, and allows to retrieve reads in any regionsswiftly.
Samtools is designed to work on a stream. It regards an input file ‘-’ as the standard input (stdin) and an output file ‘-’ as the standard output (stdout). Several commands can thus be combined with Unix pipes. Samtools always
output warning and error messages to the standard error output (stderr).
Samtools is also able to open a BAM (not SAM) file on a remote FTP or HTTP server if the BAM file name starts with ‘ftp://’ or ‘http://’. Samtools checks the current working directory for the index file and will download the index
upon absence. Samtools does not retrieve the entire alignment file unless it is asked to do so.
SAMTOOLS COMMANDS AND OPTIONS
view | samtools view [-bchuHS] [-t in.refList] [-o output] [-f reqFlag] [-F skipFlag] [-q minMapQ] [-l library] [-r readGroup] [-R rgFile] <in.bam>|<in.sam> [region1 [...]] Extract/print all or sub alignments in SAM or BAM format. If no region is specified, all the alignments will be printed; otherwise only alignments overlapping the specified regions will be output. An alignment may be given multiple times if it is overlapping several regions. A region can be presented, for example, in the following format: ‘chr2’ (the whole chr2), ‘chr2:1000000’ (region starting from 1,000,000bp) or ‘chr2:1,000,000-2,000,000’ (region between 1,000,000 and 2,000,000bp including the end points). The coordinate is 1-based. OPTIONS:
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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 alignment 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 [-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. In the pileup format (without -uor-g), each line represents a genomic position, consisting of chromosome name, coordinate, reference base, read bases, read qualities and alignment mapping qualities. Information on match, mismatch, indel, strand, mapping quality and start and end of a read are all encoded at the read base column. At this column, a dot stands for a match to the reference base on the forward strand, a comma for a match on the reverse strand, a ’>’ or ’<’ for a reference skip, ‘ACGTN’ for a mismatch on the forward strand and ‘acgtn’ for a mismatch on the reverse strand. A pattern ‘\+[0-9]+[ACGTNacgtn]+’ indicates there is an insertion between this reference position and the next reference position. The length of the insertion is given by the integer in the pattern, followed by the inserted sequence. Similarly, a pattern ‘-[0-9]+[ACGTNacgtn]+’ represents a deletion from the reference. The deleted bases will be presented as ‘*’ in the following lines. Also at the read base column, a symbol ‘^’ marks the start of a read. The ASCII of the character following ‘^’ minus 33 gives the mapping quality. A symbol ‘$’ marks the end of a read segment. Input Options:
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reheader | samtools reheader <in.header.sam> <in.bam> Replace the header in in.bam with the header in in.header.sam. This command is much faster than replacing the header with a BAM->SAM->BAM conversion. | ||||||||||||||||||||||||||||||||||||||||||||||||
cat | samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ] Concatenate BAMs. The sequence dictionary of each input BAM must be identical, although this command does not check this. This command uses a similar trick toreheader which enables fast BAM concatenation. | ||||||||||||||||||||||||||||||||||||||||||||||||
sort | samtools sort [-no] [-m maxMem] <in.bam> <out.prefix> Sort alignments by leftmost coordinates. File <out.prefix>.bam will be created. This command may also create temporary files <out.prefix>.%d.bam when the whole alignment cannot be fitted into memory (controlled by option -m). OPTIONS:
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merge | samtools merge [-nur1f] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...] Merge multiple sorted alignments. The header reference lists of all the input BAM files, and the @SQ headers of inh.sam, if any, must all refer to the same set of reference sequences. The header reference list and (unless overridden by -h) ‘@’ headers of in1.bam will be copied to out.bam, and the headers of other files will be ignored. OPTIONS:
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index | samtools index <aln.bam> Index sorted alignment for fast random access. Index file <aln.bam>.bai will be created. | ||||||||||||||||||||||||||||||||||||||||||||||||
idxstats | samtools idxstats <aln.bam> Retrieve and print stats in the index file. The output is TAB delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads. | ||||||||||||||||||||||||||||||||||||||||||||||||
faidx | samtools faidx <ref.fasta> [region1 [...]] Index reference sequence in the FASTA format or extract subsequence from indexed reference sequence. If no region is specified, faidx will index the file and create<ref.fasta>.fai on the disk. If regions are speficified, the subsequences will be retrieved and printed to stdout in the FASTA format. The input file can be compressed in the RAZF format. | ||||||||||||||||||||||||||||||||||||||||||||||||
fixmate | samtools fixmate <in.nameSrt.bam> <out.bam> Fill in mate coordinates, ISIZE and mate related flags from a name-sorted alignment. | ||||||||||||||||||||||||||||||||||||||||||||||||
rmdup | samtools rmdup [-sS] <input.srt.bam> <out.bam> Remove potential PCR duplicates: if multiple read pairs have identical external coordinates, only retain the pair with highest mapping quality. In the paired-end mode, this command ONLY works with FR orientation and requires ISIZE is correctly set. It does not work for unpaired reads (e.g. two ends mapped to different chromosomes or orphan reads). OPTIONS:
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calmd | samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta> Generate the MD tag. If the MD tag is already present, this command will give a warning if the MD tag generated is different from the existing tag. Output SAM by default. OPTIONS:
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targetcut | samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam> This command identifies target regions by examining the continuity of read depth, computes haploid consensus sequences of targets and outputs a SAM with each sequence corresponding to a target. When option -f is in use, BAQ will be applied. This command is only designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)]. |
phase | samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam> Call and phase heterozygous SNPs. OPTIONS:
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BCFTOOLS COMMANDS AND OPTIONS
view | bcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s listSample] [-igapSNPratio] [-t mutRate] [-p varThres] [-P prior] [-1 nGroup1] [-d minFrac] [-UnPerm] [-X permThres] [-T trioType] in.bcf [region] Convert between BCF and VCF, call variant candidates and estimate allele frequencies.
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index | bcftools index in.bcf Index sorted BCF for random access. |
cat | bcftools cat in1.bcf ["in2.bcf "[..."]]]" Concatenate BCF files. The input files are required to be sorted and have identical samples appearing in the same order. |
SAM FORMAT
Sequence Alignment/Map (SAM) format is TAB-delimited. Apart from the header lines, which are started with the ‘@’ symbol, each alignment line consists of:Col | Field | Description |
1 | QNAME | Query template/pair NAME |
2 | FLAG | bitwise FLAG |
3 | RNAME | Reference sequence NAME |
4 | POS | 1-based leftmost POSition/coordinate of clipped sequence |
5 | MAPQ | MAPping Quality (Phred-scaled) |
6 | CIAGR | extended CIGAR string |
7 | MRNM | Mate Reference sequence NaMe (‘=’ if same as RNAME) |
8 | MPOS | 1-based Mate POSistion |
9 | TLEN | inferred Template LENgth (insert size) |
10 | SEQ | query SEQuence on the same strand as the reference |
11 | QUAL | query QUALity (ASCII-33 gives the Phred base quality) |
12+ | OPT | variable OPTional fields in the format TAG:VTYPE:VALUE |
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 |
VCF FORMAT
The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:Col | Field | Description |
1 | CHROM | CHROMosome name |
2 | POS | the left-most POSition of the variant |
3 | ID | unique variant IDentifier |
4 | REF | the REFerence allele |
5 | ALT | the ALTernate allele(s), separated by comma |
6 | QUAL | variant/reference QUALity |
7 | FILTER | FILTers applied |
8 | INFO | INFOrmation related to the variant, separated by semi-colon |
9 | FORMAT | FORMAT of the genotype fields, separated by colon (optional) |
10+ | SAMPLE | SAMPLE genotypes and per-sample information (optional) |
Tag | Format | Description |
AF1 | double | Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele |
DP | int | Raw read depth (without quality filtering) |
DP4 | int[4] | # high-quality reference forward bases, ref reverse, alternate for and alt rev bases |
FQ | int | Consensus quality. Positive: sample genotypes different; negative: otherwise |
MQ | int | Root-Mean-Square mapping quality of covering reads |
PC2 | int[2] | Phred probability of AF in group1 samples being larger (,smaller) than in group2 |
PCHI2 | double | Posterior weighted chi^2 P-value between group1 and group2 samples |
PV4 | double[4] | P-value for strand bias, baseQ bias, mapQ bias and tail distance bias |
QCHI2 | int | Phred-scaled PCHI2 |
RP | int | # permutations yielding a smaller PCHI2 |
CLR | int | Phred log ratio of genotype likelihoods with and without the trio/pair constraint |
UGT | string | Most probable genotype configuration without the trio constraint |
CGT | string | Most probable configuration with the trio constraint |
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:Illumina\n@RG\tID:454\tSM:hs\tLB:454\tPL:454\n"’ > rg.txt samtools merge -rh rg.txt merged.bam ga.bam 454.bam The value in a RG tag is determined by the file name the read is coming from. In this example, in the merged.bam, reads from ga.bam will be attached RG:Z:ga, while reads from454.bam will be attached RG:Z:454. |
o | Call SNPs and short INDELs for one diploid individual: samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf bcftools view var.raw.bcf | 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 usually helps BWA-short but may not other mappers. |
o | Generate the consensus sequence for one diploid individual: samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq |
o | Call somatic mutations from a pair of samples: samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf In the output INFO field, CLR gives the Phred-log ratio between the likelihood by treating the two samples independently, and the likelihood by requiring the genotype to be identical. This CLR is effectively a score measuring the confidence of somatic calls. The higher the better. |
o | Call de novo and somatic mutations from a family trio: samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf File samples.txt should consist of three lines specifying the member and order of samples (in the order of child-father-mother). Similarly, CLR gives the Phred-log likelihood ratio with and without the trio constraint. UGT shows the most likely genotype configuration without the trio constraint, and CGT gives the most likely genotype configuration satisfying the trio constraint. |
o | Phase one individual: samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out The calmd command is used to reduce false heterozygotes around INDELs. |
o | Call SNPs and short indels for multiple diploid individuals: samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf Individuals are identified from the SM tags in the @RG header lines. Individuals can be pooled in one alignment file; one individual can also be separated into multiple files. The-P option specifies that indel candidates should be collected only from read groups with the @RG-PL tag set to ILLUMINA. Collecting indel candidates from reads sequenced by an indel-prone technology may affect the performance of indel calling. |
o | Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals: samtools mpileup -Igf ref.fa *.bam > all.bcf bcftools view -bl sites.list all.bcf > sites.bcf bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs ...... where sites.list contains the list of sites with each line consisting of the reference sequence name and position. The following bcftools commands estimate AFS by EM. |
o | Dump BAQ applied alignment for other SNP callers: samtools calmd -bAr aln.bam > aln.baq.bam It adds and corrects the NM and MD tags at the same time. The calmd command also comes with the -C option, the same as the one in pileup and mpileup. Apply if it helps. |
LIMITATIONS
o | Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c. |
o | Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan reads or ends mapped to different chromosomes). If this is a concern, please use Picard’s MarkDuplicate which correctly handles these cases, although a little slower. |
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. John Marshall and PetrDanecek contribute to the source code and various people from the 1000 Genomes Project have contributed to the SAM format specification.
SEE ALSO
Samtools website: <http://samtools.sourceforge.net>samtools-0.1.17 | samtools (1) | 05 July 2011 |
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