1 .TH samtools 1 "1 March 2011" "samtools-0.1.13" "Bioinformatics tools"
4 samtools - Utilities for the Sequence Alignment/Map (SAM) format
7 samtools view -bt ref_list.txt -o aln.bam aln.sam.gz
9 samtools sort aln.bam aln.sorted
11 samtools index aln.sorted.bam
13 samtools idxstats aln.sorted.bam
15 samtools view aln.sorted.bam chr2:20,100,000-20,200,000
17 samtools merge out.bam in1.bam in2.bam in3.bam
19 samtools faidx ref.fasta
21 samtools pileup -vcf ref.fasta aln.sorted.bam
23 samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
25 samtools tview aln.sorted.bam ref.fasta
29 Samtools is a set of utilities that manipulate alignments in the BAM
30 format. It imports from and exports to the SAM (Sequence Alignment/Map)
31 format, does sorting, merging and indexing, and allows to retrieve reads
32 in any regions swiftly.
34 Samtools is designed to work on a stream. It regards an input file `-'
35 as the standard input (stdin) and an output file `-' as the standard
36 output (stdout). Several commands can thus be combined with Unix
37 pipes. Samtools always output warning and error messages to the standard
38 error output (stderr).
40 Samtools is also able to open a BAM (not SAM) file on a remote FTP or
41 HTTP server if the BAM file name starts with `ftp://' or `http://'.
42 Samtools checks the current working directory for the index file and
43 will download the index upon absence. Samtools does not retrieve the
44 entire alignment file unless it is asked to do so.
46 .SH COMMANDS AND OPTIONS
50 samtools view [-bchuHS] [-t in.refList] [-o output] [-f reqFlag] [-F
51 skipFlag] [-q minMapQ] [-l library] [-r readGroup] [-R rgFile] <in.bam>|<in.sam> [region1 [...]]
53 Extract/print all or sub alignments in SAM or BAM format. If no region
54 is specified, all the alignments will be printed; otherwise only
55 alignments overlapping the specified regions will be output. An
56 alignment may be given multiple times if it is overlapping several
57 regions. A region can be presented, for example, in the following
58 format: `chr2' (the whole chr2), `chr2:1000000' (region starting from
59 1,000,000bp) or `chr2:1,000,000-2,000,000' (region between 1,000,000 and
60 2,000,000bp including the end points). The coordinate is 1-based.
66 Output in the BAM format.
69 Only output alignments with all bits in INT present in the FLAG
70 field. INT can be in hex in the format of /^0x[0-9A-F]+/ [0]
73 Skip alignments with bits present in INT [0]
76 Include the header in the output.
79 Output the header only.
82 Only output reads in library STR [null]
88 Skip alignments with MAPQ smaller than INT [0]
91 Only output reads in read group STR [null]
94 Output reads in read groups listed in
99 Input is in SAM. If @SQ header lines are absent, the
104 Instead of printing the alignments, only count them and print the
105 total number. All filter options, such as
110 , are taken into account.
113 This file is TAB-delimited. Each line must contain the reference name
114 and the length of the reference, one line for each distinct reference;
115 additional fields are ignored. This file also defines the order of the
116 reference sequences in sorting. If you run `samtools faidx <ref.fa>',
117 the resultant index file
124 Output uncompressed BAM. This option saves time spent on
125 compression/decomprssion and is thus preferred when the output is piped
126 to another samtools command.
131 samtools tview <in.sorted.bam> [ref.fasta]
133 Text alignment viewer (based on the ncurses library). In the viewer,
134 press `?' for help and press `g' to check the alignment start from a
135 region in the format like `chr10:10,000,000' or `=10,000,000' when
136 viewing the same reference sequence.
140 samtools mpileup [-EBug] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-M capMapQ] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]
142 Generate BCF or pileup for one or multiple BAM files. Alignment records
143 are grouped by sample identifiers in @RG header lines. If sample
144 identifiers are absent, each input file is regarded as one sample.
150 Disable probabilistic realignment for the computation of base alignment
151 quality (BAQ). BAQ is the Phred-scaled probability of a read base being
152 misaligned. Applying this option greatly helps to reduce false SNPs
153 caused by misalignments.
156 Coefficient for downgrading mapping quality for reads containing
157 excessive mismatches. Given a read with a phred-scaled probability q of
158 being generated from the mapped position, the new mapping quality is
159 about sqrt((INT-q)/INT)*INT. A zero value disables this
160 functionality; if enabled, the recommended value for BWA is 50. [0]
163 Phred-scaled gap extension sequencing error probability. Reducing
165 leads to longer indels. [20]
168 Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt
169 specificity a little bit.
172 The reference file [null]
175 Compute genotype likelihoods and output them in the binary call format (BCF).
178 Coefficient for modeling homopolymer errors. Given an
181 run, the sequencing error of an indel of size
188 File containing a list of sites where pileup or BCF is outputted [null]
191 Phred-scaled gap open sequencing error probability. Reducing
193 leads to more indel calls. [40]
196 Comma dilimited list of platforms (determined by
198 from which indel candidates are obtained. It is recommended to collect
199 indel candidates from sequencing technologies that have low indel error
200 rate such as ILLUMINA. [all]
203 Minimum mapping quality for an alignment to be used [0]
206 Minimum base quality for a base to be considered [13]
209 Only generate pileup in region
216 except that the output is uncompressed BCF, which is preferred for piping.
221 samtools reheader <in.header.sam> <in.bam>
223 Replace the header in
227 This command is much faster than replacing the header with a
228 BAM->SAM->BAM conversion.
232 samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>
234 Sort alignments by leftmost coordinates. File
236 will be created. This command may also create temporary files
237 .I <out.prefix>.%d.bam
238 when the whole alignment cannot be fitted into memory (controlled by
245 Output the final alignment to the standard output.
248 Sort by read names rather than by chromosomal coordinates
251 Approximately the maximum required memory. [500000000]
256 samtools merge [-nur] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]
258 Merge multiple sorted alignments.
259 The header reference lists of all the input BAM files, and the @SQ headers of
261 if any, must all refer to the same set of reference sequences.
262 The header reference list and (unless overridden by
268 and the headers of other files will be ignored.
276 as `@' headers to be copied to
278 replacing any header lines that would otherwise be copied from
281 is actually in SAM format, though any alignment records it may contain
285 Merge files in the specified region indicated by
289 Attach an RG tag to each alignment. The tag value is inferred from file names.
292 The input alignments are sorted by read names rather than by chromosomal
296 Uncompressed BAM output
301 samtools index <aln.bam>
303 Index sorted alignment for fast random access. Index file
309 samtools idxstats <aln.bam>
311 Retrieve and print stats in the index file. The output is TAB delimited
312 with each line consisting of reference sequence name, sequence length, #
313 mapped reads and # unmapped reads.
317 samtools faidx <ref.fasta> [region1 [...]]
319 Index reference sequence in the FASTA format or extract subsequence from
320 indexed reference sequence. If no region is specified,
322 will index the file and create
324 on the disk. If regions are speficified, the subsequences will be
325 retrieved and printed to stdout in the FASTA format. The input file can
332 samtools fixmate <in.nameSrt.bam> <out.bam>
334 Fill in mate coordinates, ISIZE and mate related flags from a
335 name-sorted alignment.
339 samtools rmdup [-sS] <input.srt.bam> <out.bam>
341 Remove potential PCR duplicates: if multiple read pairs have identical
342 external coordinates, only retain the pair with highest mapping quality.
343 In the paired-end mode, this command
345 works with FR orientation and requires ISIZE is correctly set. It does
346 not work for unpaired reads (e.g. two ends mapped to different
347 chromosomes or orphan reads).
353 Remove duplicate for single-end reads. By default, the command works for
354 paired-end reads only.
357 Treat paired-end reads and single-end reads.
362 samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta>
364 Generate the MD tag. If the MD tag is already present, this command will
365 give a warning if the MD tag generated is different from the existing
366 tag. Output SAM by default.
372 When used jointly with
374 this option overwrites the original base quality.
377 Convert a the read base to = if it is identical to the aligned reference
378 base. Indel caller does not support the = bases at the moment.
381 Output uncompressed BAM
384 Output compressed BAM
387 The input is SAM with header lines
390 Coefficient to cap mapping quality of poorly mapped reads. See the
392 command for details. [0]
395 Compute the BQ tag (without -A) or cap base quality by BAQ (with -A).
398 Extended BAQ calculation. This option trades specificity for sensitivity, though the
404 samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam>
406 This command identifies target regions by examining the continuity of read depth, computes
407 haploid consensus sequences of targets and outputs a SAM with each sequence corresponding
408 to a target. When option
410 is in use, BAQ will be applied. This command is
412 designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].
417 samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam>
419 Call and phase heterozygous SNPs.
424 Drop reads with ambiguous phase.
427 Prefix of BAM output. When this option is in use, phase-0 reads will be saved in file
431 Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads
432 with switch errors will be saved in
433 .BR STR .chimeric.bam.
437 Do not attempt to fix chimeric reads.
440 Maximum length for local phasing. [13]
443 Minimum Phred-scaled LOD to call a heterozygote. [40]
446 Minimum base quality to be used in het calling. [13]
451 samtools pileup [-2sSBicv] [-f in.ref.fasta] [-t in.ref_list] [-l
452 in.site_list] [-C capMapQ] [-M maxMapQ] [-T theta] [-N nHap] [-r
453 pairDiffRate] [-m mask] [-d maxIndelDepth] [-G indelPrior]
456 Print the alignment in the pileup format. In the pileup format, each
457 line represents a genomic position, consisting of chromosome name,
458 coordinate, reference base, read bases, read qualities and alignment
459 mapping qualities. Information on match, mismatch, indel, strand,
460 mapping quality and start and end of a read are all encoded at the read
461 base column. At this column, a dot stands for a match to the reference
462 base on the forward strand, a comma for a match on the reverse strand,
463 a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward
464 strand and `acgtn' for a mismatch on the reverse strand. A pattern
465 `\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this
466 reference position and the next reference position. The length of the
467 insertion is given by the integer in the pattern, followed by the
468 inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+'
469 represents a deletion from the reference. The deleted bases will be
470 presented as `*' in the following lines. Also at the read base column, a
471 symbol `^' marks the start of a read. The ASCII of the character
472 following `^' minus 33 gives the mapping quality. A symbol `$' marks the
473 end of a read segment.
477 is applied, the consensus base, Phred-scaled consensus quality, SNP
478 quality (i.e. the Phred-scaled probability of the consensus being
479 identical to the reference) and root mean square (RMS) mapping quality
480 of the reads covering the site will be inserted between the `reference
481 base' and the `read bases' columns. An indel occupies an additional
482 line. Each indel line consists of chromosome name, coordinate, a star,
483 the genotype, consensus quality, SNP quality, RMS mapping quality, #
484 covering reads, the first alllele, the second allele, # reads supporting
485 the first allele, # reads supporting the second allele and # reads
486 containing indels different from the top two alleles.
489 Since 0.1.10, the `pileup' command is deprecated by `mpileup'.
495 Disable the BAQ computation. See the
500 Call the consensus sequence. Options
501 .BR -T ", " -N ", " -I " and " -r
502 are only effective when
507 Coefficient for downgrading the mapping quality of poorly mapped
510 command for details. [0]
515 reads in the pileup for indel calling for speed up. Zero for unlimited. [1024]
518 The reference sequence in the FASTA format. Index file
524 Generate genotype likelihood in the binary GLFv3 format. This option
525 suppresses -c, -i and -s. This option is deprecated by the
530 Only output pileup lines containing indels.
533 Phred probability of an indel in sequencing/prep. [40]
536 List of sites at which pileup is output. This file is space
537 delimited. The first two columns are required to be chromosome and
538 1-based coordinate. Additional columns are ignored. It is
539 recommended to use option
542 Filter reads with flag containing bits in
547 Cap mapping quality at INT [60]
550 Number of haplotypes in the sample (>=2) [2]
553 Expected fraction of differences between a pair of haplotypes [0.001]
556 Print the mapping quality as the last column. This option makes the
557 output easier to parse, although this format is not space efficient.
560 The input file is in SAM.
563 List of reference names ane sequence lengths, in the format described
566 command. If this option is present, samtools assumes the input
568 is in SAM format; otherwise it assumes in BAM format.
572 as in the default format we may not know the mapping quality.
575 The theta parameter (error dependency coefficient) in the maq consensus
581 SAM is TAB-delimited. Apart from the header lines, which are started
582 with the `@' symbol, each alignment line consists of:
588 Col Field Description
590 1 QNAME Query (pair) NAME
592 3 RNAME Reference sequence NAME
593 4 POS 1-based leftmost POSition/coordinate of clipped sequence
594 5 MAPQ MAPping Quality (Phred-scaled)
595 6 CIAGR extended CIGAR string
596 7 MRNM Mate Reference sequence NaMe (`=' if same as RNAME)
597 8 MPOS 1-based Mate POSistion
598 9 ISIZE Inferred insert SIZE
599 10 SEQ query SEQuence on the same strand as the reference
600 11 QUAL query QUALity (ASCII-33 gives the Phred base quality)
601 12 OPT variable OPTional fields in the format TAG:VTYPE:VALUE
605 Each bit in the FLAG field is defined as:
613 0x0001 p the read is paired in sequencing
614 0x0002 P the read is mapped in a proper pair
615 0x0004 u the query sequence itself is unmapped
616 0x0008 U the mate is unmapped
617 0x0010 r strand of the query (1 for reverse)
618 0x0020 R strand of the mate
619 0x0040 1 the read is the first read in a pair
620 0x0080 2 the read is the second read in a pair
621 0x0100 s the alignment is not primary
622 0x0200 f the read fails platform/vendor quality checks
623 0x0400 d the read is either a PCR or an optical duplicate
628 Import SAM to BAM when
630 lines are present in the header:
632 samtools view -bS aln.sam > aln.bam
638 samtools faidx ref.fa
639 samtools view -bt ref.fa.fai aln.sam > aln.bam
643 is generated automatically by the
650 tag while merging sorted alignments:
652 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
653 samtools merge -rh rg.txt merged.bam ga.bam 454.bam
657 tag is determined by the file name the read is coming from. In this
670 Call SNPs and short indels for one diploid individual:
672 samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
673 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 > var.flt.vcf
677 option of varFilter controls the maximum read depth, which should be
678 adjusted to about twice the average read depth. One may consider to add
682 if mapping quality is overestimated for reads containing excessive
683 mismatches. Applying this option usually helps
685 but may not other mappers.
688 Generate the consensus sequence for one diploid individual:
690 samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq
693 Phase one individual:
695 samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out
699 command is used to reduce false heterozygotes around INDELs.
702 Call SNPs and short indels for multiple diploid individuals:
704 samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf
705 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf
707 Individuals are identified from the
711 header lines. Individuals can be pooled in one alignment file; one
712 individual can also be separated into multiple files. The
714 option specifies that indel candidates should be collected only from
719 Collecting indel candidates from reads sequenced by an indel-prone
720 technology may affect the performance of indel calling.
723 Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals:
725 samtools mpileup -Igf ref.fa *.bam > all.bcf
726 bcftools view -bl sites.list all.bcf > sites.bcf
727 bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
728 bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
729 bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
734 contains the list of sites with each line consisting of the reference
735 sequence name and position. The following
737 commands estimate AFS by EM.
740 Dump BAQ applied alignment for other SNP callers:
742 samtools calmd -bAr aln.bam > aln.baq.bam
744 It adds and corrects the
748 tags at the same time. The
750 command also comes with the
752 option, the same as the one in
761 Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
763 In merging, the input files are required to have the same number of
764 reference sequences. The requirement can be relaxed. In addition,
765 merging does not reconstruct the header dictionaries
766 automatically. Endusers have to provide the correct header. Picard is
769 Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan
770 reads or ends mapped to different chromosomes). If this is a concern,
771 please use Picard's MarkDuplicate which correctly handles these cases,
772 although a little slower.
776 Heng Li from the Sanger Institute wrote the C version of samtools. Bob
777 Handsaker from the Broad Institute implemented the BGZF library and Jue
778 Ruan from Beijing Genomics Institute wrote the RAZF library. John
779 Marshall and Petr Danecek contribute to the source code and various
780 people from the 1000 Genomes Project have contributed to the SAM format
785 Samtools website: <http://samtools.sourceforge.net>
projects / samtools.git / blob