1 .TH samtools 1 "2 September 2011" "samtools-0.1.18" "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 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.
143 samtools tview <in.sorted.bam> [ref.fasta]
145 Text alignment viewer (based on the ncurses library). In the viewer,
146 press `?' for help and press `g' to check the alignment start from a
147 region in the format like `chr10:10,000,000' or `=10,000,000' when
148 viewing the same reference sequence.
172 Generate BCF or pileup for one or multiple BAM files. Alignment records
173 are grouped by sample identifiers in @RG header lines. If sample
174 identifiers are absent, each input file is regarded as one sample.
176 In the pileup format (without
179 line represents a genomic position, consisting of chromosome name,
180 coordinate, reference base, read bases, read qualities and alignment
181 mapping qualities. Information on match, mismatch, indel, strand,
182 mapping quality and start and end of a read are all encoded at the read
183 base column. At this column, a dot stands for a match to the reference
184 base on the forward strand, a comma for a match on the reverse strand,
185 a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward
186 strand and `acgtn' for a mismatch on the reverse strand. A pattern
187 `\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this
188 reference position and the next reference position. The length of the
189 insertion is given by the integer in the pattern, followed by the
190 inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+'
191 represents a deletion from the reference. The deleted bases will be
192 presented as `*' in the following lines. Also at the read base column, a
193 symbol `^' marks the start of a read. The ASCII of the character
194 following `^' minus 33 gives the mapping quality. A symbol `$' marks the
195 end of a read segment.
201 Assume the quality is in the Illumina 1.3+ encoding.
203 Do not skip anomalous read pairs in variant calling.
206 Disable probabilistic realignment for the computation of base alignment
207 quality (BAQ). BAQ is the Phred-scaled probability of a read base being
208 misaligned. Applying this option greatly helps to reduce false SNPs
209 caused by misalignments.
212 List of input BAM files, one file per line [null]
215 Coefficient for downgrading mapping quality for reads containing
216 excessive mismatches. Given a read with a phred-scaled probability q of
217 being generated from the mapped position, the new mapping quality is
218 about sqrt((INT-q)/INT)*INT. A zero value disables this
219 functionality; if enabled, the recommended value for BWA is 50. [0]
222 At a position, read maximally
224 reads per input BAM. [250]
227 Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt
228 specificity a little bit.
233 reference file in the FASTA format. The file can be optionally compressed by
238 BED or position list file containing a list of regions or sites where pileup or BCF should be generated [null]
241 Minimum mapping quality for an alignment to be used [0]
244 Minimum base quality for a base to be considered [13]
247 Only generate pileup in region
255 Output per-sample read depth
258 Compute genotype likelihoods and output them in the binary call format (BCF).
261 Output per-sample Phred-scaled strand bias P-value
266 except that the output is uncompressed BCF, which is preferred for piping.
269 .B Options for Genotype Likelihood Computation (for -g or -u):
273 Phred-scaled gap extension sequencing error probability. Reducing
275 leads to longer indels. [20]
278 Coefficient for modeling homopolymer errors. Given an
281 run, the sequencing error of an indel of size
288 Do not perform INDEL calling
291 Skip INDEL calling if the average per-sample depth is above
296 Phred-scaled gap open sequencing error probability. Reducing
298 leads to more indel calls. [40]
301 Comma dilimited list of platforms (determined by
303 from which indel candidates are obtained. It is recommended to collect
304 indel candidates from sequencing technologies that have low indel error
305 rate such as ILLUMINA. [all]
310 samtools reheader <in.header.sam> <in.bam>
312 Replace the header in
316 This command is much faster than replacing the header with a
317 BAM->SAM->BAM conversion.
321 samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ]
323 Concatenate BAMs. The sequence dictionary of each input BAM must be identical,
324 although this command does not check this. This command uses a similar trick
327 which enables fast BAM concatenation.
331 samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>
333 Sort alignments by leftmost coordinates. File
335 will be created. This command may also create temporary files
336 .I <out.prefix>.%d.bam
337 when the whole alignment cannot be fitted into memory (controlled by
344 Output the final alignment to the standard output.
347 Sort by read names rather than by chromosomal coordinates
350 Approximately the maximum required memory. [500000000]
355 samtools merge [-nur1f] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]
357 Merge multiple sorted alignments.
358 The header reference lists of all the input BAM files, and the @SQ headers of
360 if any, must all refer to the same set of reference sequences.
361 The header reference list and (unless overridden by
367 and the headers of other files will be ignored.
373 Use zlib compression level 1 to comrpess the output
376 Force to overwrite the output file if present.
381 as `@' headers to be copied to
383 replacing any header lines that would otherwise be copied from
386 is actually in SAM format, though any alignment records it may contain
390 The input alignments are sorted by read names rather than by chromosomal
394 Merge files in the specified region indicated by
399 Attach an RG tag to each alignment. The tag value is inferred from file names.
402 Uncompressed BAM output
407 samtools index <aln.bam>
409 Index sorted alignment for fast random access. Index file
415 samtools idxstats <aln.bam>
417 Retrieve and print stats in the index file. The output is TAB delimited
418 with each line consisting of reference sequence name, sequence length, #
419 mapped reads and # unmapped reads.
423 samtools faidx <ref.fasta> [region1 [...]]
425 Index reference sequence in the FASTA format or extract subsequence from
426 indexed reference sequence. If no region is specified,
428 will index the file and create
430 on the disk. If regions are speficified, the subsequences will be
431 retrieved and printed to stdout in the FASTA format. The input file can
438 samtools fixmate <in.nameSrt.bam> <out.bam>
440 Fill in mate coordinates, ISIZE and mate related flags from a
441 name-sorted alignment.
445 samtools rmdup [-sS] <input.srt.bam> <out.bam>
447 Remove potential PCR duplicates: if multiple read pairs have identical
448 external coordinates, only retain the pair with highest mapping quality.
449 In the paired-end mode, this command
451 works with FR orientation and requires ISIZE is correctly set. It does
452 not work for unpaired reads (e.g. two ends mapped to different
453 chromosomes or orphan reads).
459 Remove duplicate for single-end reads. By default, the command works for
460 paired-end reads only.
463 Treat paired-end reads and single-end reads.
468 samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta>
470 Generate the MD tag. If the MD tag is already present, this command will
471 give a warning if the MD tag generated is different from the existing
472 tag. Output SAM by default.
478 When used jointly with
480 this option overwrites the original base quality.
483 Convert a the read base to = if it is identical to the aligned reference
484 base. Indel caller does not support the = bases at the moment.
487 Output uncompressed BAM
490 Output compressed BAM
493 The input is SAM with header lines
496 Coefficient to cap mapping quality of poorly mapped reads. See the
498 command for details. [0]
501 Compute the BQ tag (without -A) or cap base quality by BAQ (with -A).
504 Extended BAQ calculation. This option trades specificity for sensitivity, though the
510 samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam>
512 This command identifies target regions by examining the continuity of read depth, computes
513 haploid consensus sequences of targets and outputs a SAM with each sequence corresponding
514 to a target. When option
516 is in use, BAQ will be applied. This command is
518 designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].
523 samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam>
525 Call and phase heterozygous SNPs.
530 Drop reads with ambiguous phase.
533 Prefix of BAM output. When this option is in use, phase-0 reads will be saved in file
537 Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads
538 with switch errors will be saved in
539 .BR STR .chimeric.bam.
543 Do not attempt to fix chimeric reads.
546 Maximum length for local phasing. [13]
549 Minimum Phred-scaled LOD to call a heterozygote. [40]
552 Minimum base quality to be used in het calling. [13]
555 .SH BCFTOOLS COMMANDS AND OPTIONS
560 .RB [ \-AbFGNQSucgv ]
588 Convert between BCF and VCF, call variant candidates and estimate allele
593 .B Input/Output Options:
596 Retain all possible alternate alleles at variant sites. By default, the view
597 command discards unlikely alleles.
600 Output in the BCF format. The default is VCF.
603 Sequence dictionary (list of chromosome names) for VCF->BCF conversion [null]
606 Indicate PL is generated by r921 or before (ordering is different).
609 Suppress all individual genotype information.
612 List of sites at which information are outputted [all sites]
615 Skip sites where the REF field is not A/C/G/T
618 Output the QCALL likelihood format
621 List of samples to use. The first column in the input gives the sample names
622 and the second gives the ploidy, which can only be 1 or 2. When the 2nd column
623 is absent, the sample ploidy is assumed to be 2. In the output, the ordering of
624 samples will be identical to the one in
629 The input is VCF instead of BCF.
632 Uncompressed BCF output (force -b).
634 .B Consensus/Variant Calling Options:
637 Call variants using Bayesian inference. This option automatically invokes option
643 is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0]
646 Perform max-likelihood inference only, including estimating the site allele frequency,
647 testing Hardy-Weinberg equlibrium and testing associations with LRT.
650 Call per-sample genotypes at variant sites (force -c)
653 Ratio of INDEL-to-SNP mutation rate [0.15]
656 A site is considered to be a variant if P(ref|D)<FLOAT [0.5]
659 Prior or initial allele frequency spectrum. If STR can be
663 or the file consisting of error output from a previous variant calling
667 Scaled muttion rate for variant calling [0.001]
670 Enable pair/trio calling. For trio calling, option
672 is usually needed to be applied to configure the trio members and their ordering.
673 In the file supplied to the option
675 the first sample must be the child, the second the father and the third the mother.
678 are `pair', `trioauto', `trioxd' and `trioxs', where `pair' calls differences between two input samples, and `trioxd' (`trioxs') specifies that the input
679 is from the X chromosome non-PAR regions and the child is a female (male). [null]
682 Output variant sites only (force -c)
684 .B Contrast Calling and Association Test Options:
687 Number of group-1 samples. This option is used for dividing the samples into
688 two groups for contrast SNP calling or association test.
689 When this option is in use, the following VCF INFO will be outputted:
690 PC2, PCHI2 and QCHI2. [0]
693 Number of permutations for association test (effective only with
698 Only perform permutations for P(chi^2)<FLOAT (effective only with
708 Index sorted BCF for random access.
715 .RI [ "in2.bcf " [ ... "]]]"
717 Concatenate BCF files. The input files are required to be sorted and
718 have identical samples appearing in the same order.
722 Sequence Alignment/Map (SAM) format is TAB-delimited. Apart from the header lines, which are started
723 with the `@' symbol, each alignment line consists of:
729 Col Field Description
731 1 QNAME Query template/pair NAME
733 3 RNAME Reference sequence NAME
734 4 POS 1-based leftmost POSition/coordinate of clipped sequence
735 5 MAPQ MAPping Quality (Phred-scaled)
736 6 CIAGR extended CIGAR string
737 7 MRNM Mate Reference sequence NaMe (`=' if same as RNAME)
738 8 MPOS 1-based Mate POSistion
739 9 TLEN inferred Template LENgth (insert size)
740 10 SEQ query SEQuence on the same strand as the reference
741 11 QUAL query QUALity (ASCII-33 gives the Phred base quality)
742 12+ OPT variable OPTional fields in the format TAG:VTYPE:VALUE
746 Each bit in the FLAG field is defined as:
754 0x0001 p the read is paired in sequencing
755 0x0002 P the read is mapped in a proper pair
756 0x0004 u the query sequence itself is unmapped
757 0x0008 U the mate is unmapped
758 0x0010 r strand of the query (1 for reverse)
759 0x0020 R strand of the mate
760 0x0040 1 the read is the first read in a pair
761 0x0080 2 the read is the second read in a pair
762 0x0100 s the alignment is not primary
763 0x0200 f the read fails platform/vendor quality checks
764 0x0400 d the read is either a PCR or an optical duplicate
767 where the second column gives the string representation of the FLAG field.
771 The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:
776 Col Field Description
778 1 CHROM CHROMosome name
779 2 POS the left-most POSition of the variant
780 3 ID unique variant IDentifier
781 4 REF the REFerence allele
782 5 ALT the ALTernate allele(s), separated by comma
783 6 QUAL variant/reference QUALity
784 7 FILTER FILTers applied
785 8 INFO INFOrmation related to the variant, separated by semi-colon
786 9 FORMAT FORMAT of the genotype fields, separated by colon (optional)
787 10+ SAMPLE SAMPLE genotypes and per-sample information (optional)
791 The following table gives the
793 tags used by samtools and bcftools.
799 Tag Format Description
801 AF1 double Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele
802 DP int Raw read depth (without quality filtering)
803 DP4 int[4] # high-quality reference forward bases, ref reverse, alternate for and alt rev bases
804 FQ int Consensus quality. Positive: sample genotypes different; negative: otherwise
805 MQ int Root-Mean-Square mapping quality of covering reads
806 PC2 int[2] Phred probability of AF in group1 samples being larger (,smaller) than in group2
807 PCHI2 double Posterior weighted chi^2 P-value between group1 and group2 samples
808 PV4 double[4] P-value for strand bias, baseQ bias, mapQ bias and tail distance bias
809 QCHI2 int Phred-scaled PCHI2
810 RP int # permutations yielding a smaller PCHI2
811 CLR int Phred log ratio of genotype likelihoods with and without the trio/pair constraint
812 UGT string Most probable genotype configuration without the trio constraint
813 CGT string Most probable configuration with the trio constraint
818 Import SAM to BAM when
820 lines are present in the header:
822 samtools view -bS aln.sam > aln.bam
828 samtools faidx ref.fa
829 samtools view -bt ref.fa.fai aln.sam > aln.bam
833 is generated automatically by the
840 tag while merging sorted alignments:
842 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
843 samtools merge -rh rg.txt merged.bam ga.bam 454.bam
847 tag is determined by the file name the read is coming from. In this
860 Call SNPs and short INDELs for one diploid individual:
862 samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
863 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 > var.flt.vcf
867 option of varFilter controls the maximum read depth, which should be
868 adjusted to about twice the average read depth. One may consider to add
872 if mapping quality is overestimated for reads containing excessive
873 mismatches. Applying this option usually helps
875 but may not other mappers.
878 Generate the consensus sequence for one diploid individual:
880 samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq
883 Call somatic mutations from a pair of samples:
885 samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf
887 In the output INFO field,
889 gives the Phred-log ratio between the likelihood by treating the
890 two samples independently, and the likelihood by requiring the genotype to be identical.
893 is effectively a score measuring the confidence of somatic calls. The higher the better.
896 Call de novo and somatic mutations from a family trio:
898 samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf
902 should consist of three lines specifying the member and order of samples (in the order of child-father-mother).
905 gives the Phred-log likelihood ratio with and without the trio constraint.
907 shows the most likely genotype configuration without the trio constraint, and
909 gives the most likely genotype configuration satisfying the trio constraint.
912 Phase one individual:
914 samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out
918 command is used to reduce false heterozygotes around INDELs.
921 Call SNPs and short indels for multiple diploid individuals:
923 samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf
924 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf
926 Individuals are identified from the
930 header lines. Individuals can be pooled in one alignment file; one
931 individual can also be separated into multiple files. The
933 option specifies that indel candidates should be collected only from
938 Collecting indel candidates from reads sequenced by an indel-prone
939 technology may affect the performance of indel calling.
942 Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals:
944 samtools mpileup -Igf ref.fa *.bam > all.bcf
945 bcftools view -bl sites.list all.bcf > sites.bcf
946 bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
947 bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
948 bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
953 contains the list of sites with each line consisting of the reference
954 sequence name and position. The following
956 commands estimate AFS by EM.
959 Dump BAQ applied alignment for other SNP callers:
961 samtools calmd -bAr aln.bam > aln.baq.bam
963 It adds and corrects the
967 tags at the same time. The
969 command also comes with the
971 option, the same as the one in
980 Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
982 Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan
983 reads or ends mapped to different chromosomes). If this is a concern,
984 please use Picard's MarkDuplicate which correctly handles these cases,
985 although a little slower.
989 Heng Li from the Sanger Institute wrote the C version of samtools. Bob
990 Handsaker from the Broad Institute implemented the BGZF library and Jue
991 Ruan from Beijing Genomics Institute wrote the RAZF library. John
992 Marshall and Petr Danecek contribute to the source code and various
993 people from the 1000 Genomes Project have contributed to the SAM format
998 Samtools website: <http://samtools.sourceforge.net>
projects / samtools.git / blob