1 .TH samtools 1 "15 March 2013" "samtools-0.1.19" "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 -Nvm0.99 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 Fraction of templates/pairs to subsample; the integer part is treated as the
108 seed for the random number generator [-1]
111 Input is in SAM. If @SQ header lines are absent, the
116 Instead of printing the alignments, only count them and print the
117 total number. All filter options, such as
122 , are taken into account.
125 This file is TAB-delimited. Each line must contain the reference name
126 and the length of the reference, one line for each distinct reference;
127 additional fields are ignored. This file also defines the order of the
128 reference sequences in sorting. If you run `samtools faidx <ref.fa>',
129 the resultant index file
136 Output uncompressed BAM. This option saves time spent on
137 compression/decomprssion and is thus preferred when the output is piped
138 to another samtools command.
153 Text alignment viewer (based on the ncurses library). In the viewer,
154 press `?' for help and press `g' to check the alignment start from a
155 region in the format like `chr10:10,000,000' or `=10,000,000' when
156 viewing the same reference sequence.
162 Output as (H)tml or (C)urses or (T)ext
165 Go directly to this position
168 Display only reads from this sample or read group
193 Generate BCF or pileup for one or multiple BAM files. Alignment records
194 are grouped by sample identifiers in @RG header lines. If sample
195 identifiers are absent, each input file is regarded as one sample.
197 In the pileup format (without
200 line represents a genomic position, consisting of chromosome name,
201 coordinate, reference base, read bases, read qualities and alignment
202 mapping qualities. Information on match, mismatch, indel, strand,
203 mapping quality and start and end of a read are all encoded at the read
204 base column. At this column, a dot stands for a match to the reference
205 base on the forward strand, a comma for a match on the reverse strand,
206 a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward
207 strand and `acgtn' for a mismatch on the reverse strand. A pattern
208 `\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this
209 reference position and the next reference position. The length of the
210 insertion is given by the integer in the pattern, followed by the
211 inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+'
212 represents a deletion from the reference. The deleted bases will be
213 presented as `*' in the following lines. Also at the read base column, a
214 symbol `^' marks the start of a read. The ASCII of the character
215 following `^' minus 33 gives the mapping quality. A symbol `$' marks the
216 end of a read segment.
222 Assume the quality is in the Illumina 1.3+ encoding.
224 Do not skip anomalous read pairs in variant calling.
227 Disable probabilistic realignment for the computation of base alignment
228 quality (BAQ). BAQ is the Phred-scaled probability of a read base being
229 misaligned. Applying this option greatly helps to reduce false SNPs
230 caused by misalignments.
233 List of input BAM files, one file per line [null]
236 Coefficient for downgrading mapping quality for reads containing
237 excessive mismatches. Given a read with a phred-scaled probability q of
238 being generated from the mapped position, the new mapping quality is
239 about sqrt((INT-q)/INT)*INT. A zero value disables this
240 functionality; if enabled, the recommended value for BWA is 50. [0]
243 At a position, read maximally
245 reads per input BAM. [250]
248 Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt
249 specificity a little bit.
254 reference file in the FASTA format. The file can be optionally compressed by
259 BED or position list file containing a list of regions or sites where pileup or BCF should be generated [null]
262 Minimum mapping quality for an alignment to be used [0]
265 Minimum base quality for a base to be considered [13]
268 Only generate pileup in region
276 Output per-sample read depth
279 Compute genotype likelihoods and output them in the binary call format (BCF).
282 Output per-sample Phred-scaled strand bias P-value
287 except that the output is uncompressed BCF, which is preferred for piping.
290 .B Options for Genotype Likelihood Computation (for -g or -u):
294 Phred-scaled gap extension sequencing error probability. Reducing
296 leads to longer indels. [20]
299 Coefficient for modeling homopolymer errors. Given an
302 run, the sequencing error of an indel of size
309 Do not perform INDEL calling
312 Skip INDEL calling if the average per-sample depth is above
317 Phred-scaled gap open sequencing error probability. Reducing
319 leads to more indel calls. [40]
322 Apply -m and -F thresholds per sample to increase sensitivity of calling.
323 By default both options are applied to reads pooled from all samples.
326 Comma dilimited list of platforms (determined by
328 from which indel candidates are obtained. It is recommended to collect
329 indel candidates from sequencing technologies that have low indel error
330 rate such as ILLUMINA. [all]
335 samtools reheader <in.header.sam> <in.bam>
337 Replace the header in
341 This command is much faster than replacing the header with a
342 BAM->SAM->BAM conversion.
346 samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ]
348 Concatenate BAMs. The sequence dictionary of each input BAM must be identical,
349 although this command does not check this. This command uses a similar trick
352 which enables fast BAM concatenation.
356 samtools sort [-nof] [-m maxMem] <in.bam> <out.prefix>
358 Sort alignments by leftmost coordinates. File
360 will be created. This command may also create temporary files
361 .I <out.prefix>.%d.bam
362 when the whole alignment cannot be fitted into memory (controlled by
369 Output the final alignment to the standard output.
372 Sort by read names rather than by chromosomal coordinates
377 as the full output path and do not append
382 Approximately the maximum required memory. [500000000]
387 samtools merge [-nur1f] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]
389 Merge multiple sorted alignments.
390 The header reference lists of all the input BAM files, and the @SQ headers of
392 if any, must all refer to the same set of reference sequences.
393 The header reference list and (unless overridden by
399 and the headers of other files will be ignored.
405 Use zlib compression level 1 to comrpess the output
408 Force to overwrite the output file if present.
413 as `@' headers to be copied to
415 replacing any header lines that would otherwise be copied from
418 is actually in SAM format, though any alignment records it may contain
422 The input alignments are sorted by read names rather than by chromosomal
426 Merge files in the specified region indicated by
431 Attach an RG tag to each alignment. The tag value is inferred from file names.
434 Uncompressed BAM output
439 samtools index <aln.bam>
441 Index sorted alignment for fast random access. Index file
447 samtools idxstats <aln.bam>
449 Retrieve and print stats in the index file. The output is TAB delimited
450 with each line consisting of reference sequence name, sequence length, #
451 mapped reads and # unmapped reads.
455 samtools faidx <ref.fasta> [region1 [...]]
457 Index reference sequence in the FASTA format or extract subsequence from
458 indexed reference sequence. If no region is specified,
460 will index the file and create
462 on the disk. If regions are speficified, the subsequences will be
463 retrieved and printed to stdout in the FASTA format. The input file can
470 samtools fixmate <in.nameSrt.bam> <out.bam>
472 Fill in mate coordinates, ISIZE and mate related flags from a
473 name-sorted alignment.
477 samtools rmdup [-sS] <input.srt.bam> <out.bam>
479 Remove potential PCR duplicates: if multiple read pairs have identical
480 external coordinates, only retain the pair with highest mapping quality.
481 In the paired-end mode, this command
483 works with FR orientation and requires ISIZE is correctly set. It does
484 not work for unpaired reads (e.g. two ends mapped to different
485 chromosomes or orphan reads).
491 Remove duplicate for single-end reads. By default, the command works for
492 paired-end reads only.
495 Treat paired-end reads and single-end reads.
500 samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta>
502 Generate the MD tag. If the MD tag is already present, this command will
503 give a warning if the MD tag generated is different from the existing
504 tag. Output SAM by default.
510 When used jointly with
512 this option overwrites the original base quality.
515 Convert a the read base to = if it is identical to the aligned reference
516 base. Indel caller does not support the = bases at the moment.
519 Output uncompressed BAM
522 Output compressed BAM
525 The input is SAM with header lines
528 Coefficient to cap mapping quality of poorly mapped reads. See the
530 command for details. [0]
533 Compute the BQ tag (without -A) or cap base quality by BAQ (with -A).
536 Extended BAQ calculation. This option trades specificity for sensitivity, though the
542 samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam>
544 This command identifies target regions by examining the continuity of read depth, computes
545 haploid consensus sequences of targets and outputs a SAM with each sequence corresponding
546 to a target. When option
548 is in use, BAQ will be applied. This command is
550 designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].
555 samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam>
557 Call and phase heterozygous SNPs.
562 Drop reads with ambiguous phase.
565 Prefix of BAM output. When this option is in use, phase-0 reads will be saved in file
569 Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads
570 with switch errors will be saved in
571 .BR STR .chimeric.bam.
575 Do not attempt to fix chimeric reads.
578 Maximum length for local phasing. [13]
581 Minimum Phred-scaled LOD to call a heterozygote. [40]
584 Minimum base quality to be used in het calling. [13]
587 .SH BCFTOOLS COMMANDS AND OPTIONS
592 .RB [ \-AbFGNQSucgv ]
622 Convert between BCF and VCF, call variant candidates and estimate allele
627 .B Input/Output Options:
630 Retain all possible alternate alleles at variant sites. By default, the view
631 command discards unlikely alleles.
634 Output in the BCF format. The default is VCF.
637 Sequence dictionary (list of chromosome names) for VCF->BCF conversion [null]
640 Indicate PL is generated by r921 or before (ordering is different).
643 Suppress all individual genotype information.
646 List of sites at which information are outputted [all sites]
649 Skip sites where the REF field is not A/C/G/T
652 Output the QCALL likelihood format
655 List of samples to use. The first column in the input gives the sample names
656 and the second gives the ploidy, which can only be 1 or 2. When the 2nd column
657 is absent, the sample ploidy is assumed to be 2. In the output, the ordering of
658 samples will be identical to the one in
663 The input is VCF instead of BCF.
666 Uncompressed BCF output (force -b).
668 .B Consensus/Variant Calling Options:
671 Call variants using Bayesian inference. This option automatically invokes option
677 is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0]
680 Perform max-likelihood inference only, including estimating the site allele frequency,
681 testing Hardy-Weinberg equlibrium and testing associations with LRT.
684 Call per-sample genotypes at variant sites (force -c)
687 Ratio of INDEL-to-SNP mutation rate [0.15]
690 New model for improved multiallelic and rare-variant calling. Another
691 ALT allele is accepted if P(chi^2) of LRT exceeds the FLOAT threshold. The
692 parameter seems robust and the actual value usually does not affect the results
693 much; a good value to use is 0.99. This is the recommended calling method. [0]
696 A site is considered to be a variant if P(ref|D)<FLOAT [0.5]
699 Prior or initial allele frequency spectrum. If STR can be
703 or the file consisting of error output from a previous variant calling
707 Scaled muttion rate for variant calling [0.001]
710 Enable pair/trio calling. For trio calling, option
712 is usually needed to be applied to configure the trio members and their ordering.
713 In the file supplied to the option
715 the first sample must be the child, the second the father and the third the mother.
718 are `pair', `trioauto', `trioxd' and `trioxs', where `pair' calls differences between two input samples, and `trioxd' (`trioxs') specifies that the input
719 is from the X chromosome non-PAR regions and the child is a female (male). [null]
722 Output variant sites only (force -c)
724 .B Contrast Calling and Association Test Options:
727 Number of group-1 samples. This option is used for dividing the samples into
728 two groups for contrast SNP calling or association test.
729 When this option is in use, the following VCF INFO will be outputted:
730 PC2, PCHI2 and QCHI2. [0]
733 Number of permutations for association test (effective only with
738 Only perform permutations for P(chi^2)<FLOAT (effective only with
748 Index sorted BCF for random access.
755 .RI [ "in2.bcf " [ ... "]]]"
757 Concatenate BCF files. The input files are required to be sorted and
758 have identical samples appearing in the same order.
762 Sequence Alignment/Map (SAM) format is TAB-delimited. Apart from the header lines, which are started
763 with the `@' symbol, each alignment line consists of:
769 Col Field Description
771 1 QNAME Query template/pair NAME
773 3 RNAME Reference sequence NAME
774 4 POS 1-based leftmost POSition/coordinate of clipped sequence
775 5 MAPQ MAPping Quality (Phred-scaled)
776 6 CIAGR extended CIGAR string
777 7 MRNM Mate Reference sequence NaMe (`=' if same as RNAME)
778 8 MPOS 1-based Mate POSistion
779 9 TLEN inferred Template LENgth (insert size)
780 10 SEQ query SEQuence on the same strand as the reference
781 11 QUAL query QUALity (ASCII-33 gives the Phred base quality)
782 12+ OPT variable OPTional fields in the format TAG:VTYPE:VALUE
786 Each bit in the FLAG field is defined as:
794 0x0001 p the read is paired in sequencing
795 0x0002 P the read is mapped in a proper pair
796 0x0004 u the query sequence itself is unmapped
797 0x0008 U the mate is unmapped
798 0x0010 r strand of the query (1 for reverse)
799 0x0020 R strand of the mate
800 0x0040 1 the read is the first read in a pair
801 0x0080 2 the read is the second read in a pair
802 0x0100 s the alignment is not primary
803 0x0200 f the read fails platform/vendor quality checks
804 0x0400 d the read is either a PCR or an optical duplicate
807 where the second column gives the string representation of the FLAG field.
811 The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:
816 Col Field Description
818 1 CHROM CHROMosome name
819 2 POS the left-most POSition of the variant
820 3 ID unique variant IDentifier
821 4 REF the REFerence allele
822 5 ALT the ALTernate allele(s), separated by comma
823 6 QUAL variant/reference QUALity
824 7 FILTER FILTers applied
825 8 INFO INFOrmation related to the variant, separated by semi-colon
826 9 FORMAT FORMAT of the genotype fields, separated by colon (optional)
827 10+ SAMPLE SAMPLE genotypes and per-sample information (optional)
831 The following table gives the
833 tags used by samtools and bcftools.
839 Tag Format Description
841 AF1 double Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele
842 DP int Raw read depth (without quality filtering)
843 DP4 int[4] # high-quality reference forward bases, ref reverse, alternate for and alt rev bases
844 FQ int Consensus quality. Positive: sample genotypes different; negative: otherwise
845 MQ int Root-Mean-Square mapping quality of covering reads
846 PC2 int[2] Phred probability of AF in group1 samples being larger (,smaller) than in group2
847 PCHI2 double Posterior weighted chi^2 P-value between group1 and group2 samples
848 PV4 double[4] P-value for strand bias, baseQ bias, mapQ bias and tail distance bias
849 QCHI2 int Phred-scaled PCHI2
850 RP int # permutations yielding a smaller PCHI2
851 CLR int Phred log ratio of genotype likelihoods with and without the trio/pair constraint
852 UGT string Most probable genotype configuration without the trio constraint
853 CGT string Most probable configuration with the trio constraint
854 VDB float Tests variant positions within reads. Intended for filtering RNA-seq artifacts around splice sites
855 RPB float Mann-Whitney rank-sum test for tail distance bias
856 HWE float Hardy-Weinberg equilibrium test, Wigginton et al., PMID: 15789306
861 Import SAM to BAM when
863 lines are present in the header:
865 samtools view -bS aln.sam > aln.bam
871 samtools faidx ref.fa
872 samtools view -bt ref.fa.fai aln.sam > aln.bam
876 is generated automatically by the
883 tag while merging sorted alignments:
885 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
886 samtools merge -rh rg.txt merged.bam ga.bam 454.bam
890 tag is determined by the file name the read is coming from. In this
903 Call SNPs and short INDELs for one diploid individual:
905 samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
906 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 > var.flt.vcf
910 option of varFilter controls the maximum read depth, which should be
911 adjusted to about twice the average read depth. One may consider to add
915 if mapping quality is overestimated for reads containing excessive
916 mismatches. Applying this option usually helps
918 but may not other mappers.
921 Generate the consensus sequence for one diploid individual:
923 samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq
926 Call somatic mutations from a pair of samples:
928 samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf
930 In the output INFO field,
932 gives the Phred-log ratio between the likelihood by treating the
933 two samples independently, and the likelihood by requiring the genotype to be identical.
936 is effectively a score measuring the confidence of somatic calls. The higher the better.
939 Call de novo and somatic mutations from a family trio:
941 samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf
945 should consist of three lines specifying the member and order of samples (in the order of child-father-mother).
948 gives the Phred-log likelihood ratio with and without the trio constraint.
950 shows the most likely genotype configuration without the trio constraint, and
952 gives the most likely genotype configuration satisfying the trio constraint.
955 Phase one individual:
957 samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out
961 command is used to reduce false heterozygotes around INDELs.
964 Call SNPs and short indels for multiple diploid individuals:
966 samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf
967 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf
969 Individuals are identified from the
973 header lines. Individuals can be pooled in one alignment file; one
974 individual can also be separated into multiple files. The
976 option specifies that indel candidates should be collected only from
981 Collecting indel candidates from reads sequenced by an indel-prone
982 technology may affect the performance of indel calling.
984 Note that there is a new calling model which can be invoked by
986 bcftools view -m0.99 ...
988 which fixes some severe limitations of the default method.
990 For filtering, best results seem to be achieved by first applying the
992 filter and then applying some machine learning approach
994 vcf-annotate -f SnpGap=n
997 Both can be found in the
1001 package (links below).
1004 Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals:
1006 samtools mpileup -Igf ref.fa *.bam > all.bcf
1007 bcftools view -bl sites.list all.bcf > sites.bcf
1008 bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
1009 bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
1010 bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
1015 contains the list of sites with each line consisting of the reference
1016 sequence name and position. The following
1018 commands estimate AFS by EM.
1021 Dump BAQ applied alignment for other SNP callers:
1023 samtools calmd -bAr aln.bam > aln.baq.bam
1025 It adds and corrects the
1029 tags at the same time. The
1031 command also comes with the
1033 option, the same as the one in
1042 Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
1044 Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan
1045 reads or ends mapped to different chromosomes). If this is a concern,
1046 please use Picard's MarkDuplicate which correctly handles these cases,
1047 although a little slower.
1051 Heng Li from the Sanger Institute wrote the C version of samtools. Bob
1052 Handsaker from the Broad Institute implemented the BGZF library and Jue
1053 Ruan from Beijing Genomics Institute wrote the RAZF library. John
1054 Marshall and Petr Danecek contribute to the source code and various
1055 people from the 1000 Genomes Project have contributed to the SAM format
1060 Samtools website: <http://samtools.sourceforge.net>
1062 Samtools latest source: <https://github.com/samtools/samtools>
1064 VCFtools website with stable link to VCF specification: <http://vcftools.sourceforge.net>
1066 HTSlib website: <https://github.com/samtools/htslib>
projects / rsem.git / blob