1 .TH samtools 1 "05 July 2011" "samtools-0.1.17" "Bioinformatics tools"
4 samtools - Utilities for the Sequence Alignment/Map (SAM) format
6 bcftools - Utilities for the Binary Call Format (BCF) and VCF
9 samtools view -bt ref_list.txt -o aln.bam aln.sam.gz
11 samtools sort aln.bam aln.sorted
13 samtools index aln.sorted.bam
15 samtools idxstats aln.sorted.bam
17 samtools view aln.sorted.bam chr2:20,100,000-20,200,000
19 samtools merge out.bam in1.bam in2.bam in3.bam
21 samtools faidx ref.fasta
23 samtools pileup -vcf ref.fasta aln.sorted.bam
25 samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
27 samtools tview aln.sorted.bam ref.fasta
31 bcftools view in.bcf chr2:100-200 > out.vcf
33 bcftools view -vc in.bcf > out.vcf 2> out.afs
37 Samtools is a set of utilities that manipulate alignments in the BAM
38 format. It imports from and exports to the SAM (Sequence Alignment/Map)
39 format, does sorting, merging and indexing, and allows to retrieve reads
40 in any regions swiftly.
42 Samtools is designed to work on a stream. It regards an input file `-'
43 as the standard input (stdin) and an output file `-' as the standard
44 output (stdout). Several commands can thus be combined with Unix
45 pipes. Samtools always output warning and error messages to the standard
46 error output (stderr).
48 Samtools is also able to open a BAM (not SAM) file on a remote FTP or
49 HTTP server if the BAM file name starts with `ftp://' or `http://'.
50 Samtools checks the current working directory for the index file and
51 will download the index upon absence. Samtools does not retrieve the
52 entire alignment file unless it is asked to do so.
54 .SH SAMTOOLS COMMANDS AND OPTIONS
58 samtools view [-bchuHS] [-t in.refList] [-o output] [-f reqFlag] [-F
59 skipFlag] [-q minMapQ] [-l library] [-r readGroup] [-R rgFile] <in.bam>|<in.sam> [region1 [...]]
61 Extract/print all or sub alignments in SAM or BAM format. If no region
62 is specified, all the alignments will be printed; otherwise only
63 alignments overlapping the specified regions will be output. An
64 alignment may be given multiple times if it is overlapping several
65 regions. A region can be presented, for example, in the following
66 format: `chr2' (the whole chr2), `chr2:1000000' (region starting from
67 1,000,000bp) or `chr2:1,000,000-2,000,000' (region between 1,000,000 and
68 2,000,000bp including the end points). The coordinate is 1-based.
74 Output in the BAM format.
77 Only output alignments with all bits in INT present in the FLAG
78 field. INT can be in hex in the format of /^0x[0-9A-F]+/ [0]
81 Skip alignments with bits present in INT [0]
84 Include the header in the output.
87 Output the header only.
90 Only output reads in library STR [null]
96 Skip alignments with MAPQ smaller than INT [0]
99 Only output reads in read group STR [null]
102 Output reads in read groups listed in
107 Input is in SAM. If @SQ header lines are absent, the
112 Instead of printing the alignments, only count them and print the
113 total number. All filter options, such as
118 , are taken into account.
121 This file is TAB-delimited. Each line must contain the reference name
122 and the length of the reference, one line for each distinct reference;
123 additional fields are ignored. This file also defines the order of the
124 reference sequences in sorting. If you run `samtools faidx <ref.fa>',
125 the resultant index file
132 Output uncompressed BAM. This option saves time spent on
133 compression/decomprssion and is thus preferred when the output is piped
134 to another samtools command.
139 samtools tview <in.sorted.bam> [ref.fasta]
141 Text alignment viewer (based on the ncurses library). In the viewer,
142 press `?' for help and press `g' to check the alignment start from a
143 region in the format like `chr10:10,000,000' or `=10,000,000' when
144 viewing the same reference sequence.
168 Generate BCF or pileup for one or multiple BAM files. Alignment records
169 are grouped by sample identifiers in @RG header lines. If sample
170 identifiers are absent, each input file is regarded as one sample.
172 In the pileup format (without
175 line represents a genomic position, consisting of chromosome name,
176 coordinate, reference base, read bases, read qualities and alignment
177 mapping qualities. Information on match, mismatch, indel, strand,
178 mapping quality and start and end of a read are all encoded at the read
179 base column. At this column, a dot stands for a match to the reference
180 base on the forward strand, a comma for a match on the reverse strand,
181 a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward
182 strand and `acgtn' for a mismatch on the reverse strand. A pattern
183 `\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this
184 reference position and the next reference position. The length of the
185 insertion is given by the integer in the pattern, followed by the
186 inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+'
187 represents a deletion from the reference. The deleted bases will be
188 presented as `*' in the following lines. Also at the read base column, a
189 symbol `^' marks the start of a read. The ASCII of the character
190 following `^' minus 33 gives the mapping quality. A symbol `$' marks the
191 end of a read segment.
197 Assume the quality is in the Illumina 1.3+ encoding.
199 Do not skip anomalous read pairs in variant calling.
202 Disable probabilistic realignment for the computation of base alignment
203 quality (BAQ). BAQ is the Phred-scaled probability of a read base being
204 misaligned. Applying this option greatly helps to reduce false SNPs
205 caused by misalignments.
208 List of input BAM files, one file per line [null]
211 Coefficient for downgrading mapping quality for reads containing
212 excessive mismatches. Given a read with a phred-scaled probability q of
213 being generated from the mapped position, the new mapping quality is
214 about sqrt((INT-q)/INT)*INT. A zero value disables this
215 functionality; if enabled, the recommended value for BWA is 50. [0]
218 At a position, read maximally
220 reads per input BAM. [250]
223 Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt
224 specificity a little bit.
229 reference file in the FASTA format. The file can be optionally compressed by
234 BED or position list file containing a list of regions or sites where pileup or BCF should be generated [null]
237 Minimum mapping quality for an alignment to be used [0]
240 Minimum base quality for a base to be considered [13]
243 Only generate pileup in region
251 Output per-sample read depth
254 Compute genotype likelihoods and output them in the binary call format (BCF).
257 Output per-sample Phred-scaled strand bias P-value
262 except that the output is uncompressed BCF, which is preferred for piping.
265 .B Options for Genotype Likelihood Computation (for -g or -u):
269 Phred-scaled gap extension sequencing error probability. Reducing
271 leads to longer indels. [20]
274 Coefficient for modeling homopolymer errors. Given an
277 run, the sequencing error of an indel of size
284 Do not perform INDEL calling
287 Skip INDEL calling if the average per-sample depth is above
292 Phred-scaled gap open sequencing error probability. Reducing
294 leads to more indel calls. [40]
297 Comma dilimited list of platforms (determined by
299 from which indel candidates are obtained. It is recommended to collect
300 indel candidates from sequencing technologies that have low indel error
301 rate such as ILLUMINA. [all]
306 samtools reheader <in.header.sam> <in.bam>
308 Replace the header in
312 This command is much faster than replacing the header with a
313 BAM->SAM->BAM conversion.
317 samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ]
319 Concatenate BAMs. The sequence dictionary of each input BAM must be identical,
320 although this command does not check this. This command uses a similar trick
323 which enables fast BAM concatenation.
327 samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>
329 Sort alignments by leftmost coordinates. File
331 will be created. This command may also create temporary files
332 .I <out.prefix>.%d.bam
333 when the whole alignment cannot be fitted into memory (controlled by
340 Output the final alignment to the standard output.
343 Sort by read names rather than by chromosomal coordinates
346 Approximately the maximum required memory. [500000000]
351 samtools merge [-nur1f] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]
353 Merge multiple sorted alignments.
354 The header reference lists of all the input BAM files, and the @SQ headers of
356 if any, must all refer to the same set of reference sequences.
357 The header reference list and (unless overridden by
363 and the headers of other files will be ignored.
369 Use zlib compression level 1 to comrpess the output
372 Force to overwrite the output file if present.
377 as `@' headers to be copied to
379 replacing any header lines that would otherwise be copied from
382 is actually in SAM format, though any alignment records it may contain
386 The input alignments are sorted by read names rather than by chromosomal
390 Merge files in the specified region indicated by
395 Attach an RG tag to each alignment. The tag value is inferred from file names.
398 Uncompressed BAM output
403 samtools index <aln.bam>
405 Index sorted alignment for fast random access. Index file
411 samtools idxstats <aln.bam>
413 Retrieve and print stats in the index file. The output is TAB delimited
414 with each line consisting of reference sequence name, sequence length, #
415 mapped reads and # unmapped reads.
419 samtools faidx <ref.fasta> [region1 [...]]
421 Index reference sequence in the FASTA format or extract subsequence from
422 indexed reference sequence. If no region is specified,
424 will index the file and create
426 on the disk. If regions are speficified, the subsequences will be
427 retrieved and printed to stdout in the FASTA format. The input file can
434 samtools fixmate <in.nameSrt.bam> <out.bam>
436 Fill in mate coordinates, ISIZE and mate related flags from a
437 name-sorted alignment.
441 samtools rmdup [-sS] <input.srt.bam> <out.bam>
443 Remove potential PCR duplicates: if multiple read pairs have identical
444 external coordinates, only retain the pair with highest mapping quality.
445 In the paired-end mode, this command
447 works with FR orientation and requires ISIZE is correctly set. It does
448 not work for unpaired reads (e.g. two ends mapped to different
449 chromosomes or orphan reads).
455 Remove duplicate for single-end reads. By default, the command works for
456 paired-end reads only.
459 Treat paired-end reads and single-end reads.
464 samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta>
466 Generate the MD tag. If the MD tag is already present, this command will
467 give a warning if the MD tag generated is different from the existing
468 tag. Output SAM by default.
474 When used jointly with
476 this option overwrites the original base quality.
479 Convert a the read base to = if it is identical to the aligned reference
480 base. Indel caller does not support the = bases at the moment.
483 Output uncompressed BAM
486 Output compressed BAM
489 The input is SAM with header lines
492 Coefficient to cap mapping quality of poorly mapped reads. See the
494 command for details. [0]
497 Compute the BQ tag (without -A) or cap base quality by BAQ (with -A).
500 Extended BAQ calculation. This option trades specificity for sensitivity, though the
506 samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam>
508 This command identifies target regions by examining the continuity of read depth, computes
509 haploid consensus sequences of targets and outputs a SAM with each sequence corresponding
510 to a target. When option
512 is in use, BAQ will be applied. This command is
514 designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].
519 samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam>
521 Call and phase heterozygous SNPs.
526 Drop reads with ambiguous phase.
529 Prefix of BAM output. When this option is in use, phase-0 reads will be saved in file
533 Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads
534 with switch errors will be saved in
535 .BR STR .chimeric.bam.
539 Do not attempt to fix chimeric reads.
542 Maximum length for local phasing. [13]
545 Minimum Phred-scaled LOD to call a heterozygote. [40]
548 Minimum base quality to be used in het calling. [13]
551 .SH BCFTOOLS COMMANDS AND OPTIONS
556 .RB [ \-AbFGNQSucgv ]
584 Convert between BCF and VCF, call variant candidates and estimate allele
589 .B Input/Output Options:
592 Retain all possible alternate alleles at variant sites. By default, the view
593 command discards unlikely alleles.
596 Output in the BCF format. The default is VCF.
599 Sequence dictionary (list of chromosome names) for VCF->BCF conversion [null]
602 Indicate PL is generated by r921 or before (ordering is different).
605 Suppress all individual genotype information.
608 List of sites at which information are outputted [all sites]
611 Skip sites where the REF field is not A/C/G/T
614 Output the QCALL likelihood format
617 List of samples to use. The first column in the input gives the sample names
618 and the second gives the ploidy, which can only be 1 or 2. When the 2nd column
619 is absent, the sample ploidy is assumed to be 2. In the output, the ordering of
620 samples will be identical to the one in
625 The input is VCF instead of BCF.
628 Uncompressed BCF output (force -b).
630 .B Consensus/Variant Calling Options:
633 Call variants using Bayesian inference. This option automatically invokes option
639 is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0]
642 Perform max-likelihood inference only, including estimating the site allele frequency,
643 testing Hardy-Weinberg equlibrium and testing associations with LRT.
646 Call per-sample genotypes at variant sites (force -c)
649 Ratio of INDEL-to-SNP mutation rate [0.15]
652 A site is considered to be a variant if P(ref|D)<FLOAT [0.5]
655 Prior or initial allele frequency spectrum. If STR can be
659 or the file consisting of error output from a previous variant calling
663 Scaled muttion rate for variant calling [0.001]
666 Enable pair/trio calling. For trio calling, option
668 is usually needed to be applied to configure the trio members and their ordering.
669 In the file supplied to the option
671 the first sample must be the child, the second the father and the third the mother.
674 are `pair', `trioauto', `trioxd' and `trioxs', where `pair' calls differences between two input samples, and `trioxd' (`trioxs') specifies that the input
675 is from the X chromosome non-PAR regions and the child is a female (male). [null]
678 Output variant sites only (force -c)
680 .B Contrast Calling and Association Test Options:
683 Number of group-1 samples. This option is used for dividing the samples into
684 two groups for contrast SNP calling or association test.
685 When this option is in use, the following VCF INFO will be outputted:
686 PC2, PCHI2 and QCHI2. [0]
689 Number of permutations for association test (effective only with
694 Only perform permutations for P(chi^2)<FLOAT (effective only with
704 Index sorted BCF for random access.
711 .RI [ "in2.bcf " [ ... "]]]"
713 Concatenate BCF files. The input files are required to be sorted and
714 have identical samples appearing in the same order.
718 Sequence Alignment/Map (SAM) format is TAB-delimited. Apart from the header lines, which are started
719 with the `@' symbol, each alignment line consists of:
725 Col Field Description
727 1 QNAME Query template/pair NAME
729 3 RNAME Reference sequence NAME
730 4 POS 1-based leftmost POSition/coordinate of clipped sequence
731 5 MAPQ MAPping Quality (Phred-scaled)
732 6 CIAGR extended CIGAR string
733 7 MRNM Mate Reference sequence NaMe (`=' if same as RNAME)
734 8 MPOS 1-based Mate POSistion
735 9 TLEN inferred Template LENgth (insert size)
736 10 SEQ query SEQuence on the same strand as the reference
737 11 QUAL query QUALity (ASCII-33 gives the Phred base quality)
738 12+ OPT variable OPTional fields in the format TAG:VTYPE:VALUE
742 Each bit in the FLAG field is defined as:
750 0x0001 p the read is paired in sequencing
751 0x0002 P the read is mapped in a proper pair
752 0x0004 u the query sequence itself is unmapped
753 0x0008 U the mate is unmapped
754 0x0010 r strand of the query (1 for reverse)
755 0x0020 R strand of the mate
756 0x0040 1 the read is the first read in a pair
757 0x0080 2 the read is the second read in a pair
758 0x0100 s the alignment is not primary
759 0x0200 f the read fails platform/vendor quality checks
760 0x0400 d the read is either a PCR or an optical duplicate
763 where the second column gives the string representation of the FLAG field.
767 The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:
772 Col Field Description
774 1 CHROM CHROMosome name
775 2 POS the left-most POSition of the variant
776 3 ID unique variant IDentifier
777 4 REF the REFerence allele
778 5 ALT the ALTernate allele(s), separated by comma
779 6 QUAL variant/reference QUALity
780 7 FILTER FILTers applied
781 8 INFO INFOrmation related to the variant, separated by semi-colon
782 9 FORMAT FORMAT of the genotype fields, separated by colon (optional)
783 10+ SAMPLE SAMPLE genotypes and per-sample information (optional)
787 The following table gives the
789 tags used by samtools and bcftools.
795 Tag Format Description
797 AF1 double Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele
798 DP int Raw read depth (without quality filtering)
799 DP4 int[4] # high-quality reference forward bases, ref reverse, alternate for and alt rev bases
800 FQ int Consensus quality. Positive: sample genotypes different; negative: otherwise
801 MQ int Root-Mean-Square mapping quality of covering reads
802 PC2 int[2] Phred probability of AF in group1 samples being larger (,smaller) than in group2
803 PCHI2 double Posterior weighted chi^2 P-value between group1 and group2 samples
804 PV4 double[4] P-value for strand bias, baseQ bias, mapQ bias and tail distance bias
805 QCHI2 int Phred-scaled PCHI2
806 RP int # permutations yielding a smaller PCHI2
807 CLR int Phred log ratio of genotype likelihoods with and without the trio/pair constraint
808 UGT string Most probable genotype configuration without the trio constraint
809 CGT string Most probable configuration with the trio constraint
814 Import SAM to BAM when
816 lines are present in the header:
818 samtools view -bS aln.sam > aln.bam
824 samtools faidx ref.fa
825 samtools view -bt ref.fa.fai aln.sam > aln.bam
829 is generated automatically by the
836 tag while merging sorted alignments:
838 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
839 samtools merge -rh rg.txt merged.bam ga.bam 454.bam
843 tag is determined by the file name the read is coming from. In this
856 Call SNPs and short INDELs for one diploid individual:
858 samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
859 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 > var.flt.vcf
863 option of varFilter controls the maximum read depth, which should be
864 adjusted to about twice the average read depth. One may consider to add
868 if mapping quality is overestimated for reads containing excessive
869 mismatches. Applying this option usually helps
871 but may not other mappers.
874 Generate the consensus sequence for one diploid individual:
876 samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq
879 Call somatic mutations from a pair of samples:
881 samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf
883 In the output INFO field,
885 gives the Phred-log ratio between the likelihood by treating the
886 two samples independently, and the likelihood by requiring the genotype to be identical.
889 is effectively a score measuring the confidence of somatic calls. The higher the better.
892 Call de novo and somatic mutations from a family trio:
894 samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf
898 should consist of three lines specifying the member and order of samples (in the order of child-father-mother).
901 gives the Phred-log likelihood ratio with and without the trio constraint.
903 shows the most likely genotype configuration without the trio constraint, and
905 gives the most likely genotype configuration satisfying the trio constraint.
908 Phase one individual:
910 samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out
914 command is used to reduce false heterozygotes around INDELs.
917 Call SNPs and short indels for multiple diploid individuals:
919 samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf
920 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf
922 Individuals are identified from the
926 header lines. Individuals can be pooled in one alignment file; one
927 individual can also be separated into multiple files. The
929 option specifies that indel candidates should be collected only from
934 Collecting indel candidates from reads sequenced by an indel-prone
935 technology may affect the performance of indel calling.
938 Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals:
940 samtools mpileup -Igf ref.fa *.bam > all.bcf
941 bcftools view -bl sites.list all.bcf > sites.bcf
942 bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
943 bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
944 bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
949 contains the list of sites with each line consisting of the reference
950 sequence name and position. The following
952 commands estimate AFS by EM.
955 Dump BAQ applied alignment for other SNP callers:
957 samtools calmd -bAr aln.bam > aln.baq.bam
959 It adds and corrects the
963 tags at the same time. The
965 command also comes with the
967 option, the same as the one in
976 Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
978 Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan
979 reads or ends mapped to different chromosomes). If this is a concern,
980 please use Picard's MarkDuplicate which correctly handles these cases,
981 although a little slower.
985 Heng Li from the Sanger Institute wrote the C version of samtools. Bob
986 Handsaker from the Broad Institute implemented the BGZF library and Jue
987 Ruan from Beijing Genomics Institute wrote the RAZF library. John
988 Marshall and Petr Danecek contribute to the source code and various
989 people from the 1000 Genomes Project have contributed to the SAM format
994 Samtools website: <http://samtools.sourceforge.net>
projects / samtools.git / blob