1 .TH samtools 1 "15 November 2010" "samtools-0.1.10" "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 [-Bug] [-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 The reference file [null]
171 Compute genotype likelihoods and output them in the binary call format (BCF).
174 Coefficient for modeling homopolymer errors. Given an
177 run, the sequencing error of an indel of size
184 File containing a list of sites where pileup or BCF is outputted [null]
187 Phred-scaled gap open sequencing error probability. Reducing
189 leads to more indel calls. [40]
192 Comma dilimited list of platforms (determined by
194 from which indel candidates are obtained. It is recommended to collect
195 indel candidates from sequencing technologies that have low indel error
196 rate such as ILLUMINA. [all]
199 Minimum mapping quality for an alignment to be used [0]
202 Minimum base quality for a base to be considered [13]
205 Only generate pileup in region
212 except that the output is uncompressed BCF, which is preferred for piping.
217 samtools reheader <in.header.sam> <in.bam>
219 Replace the header in
223 This command is much faster than replacing the header with a
224 BAM->SAM->BAM conversion.
228 samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>
230 Sort alignments by leftmost coordinates. File
232 will be created. This command may also create temporary files
233 .I <out.prefix>.%d.bam
234 when the whole alignment cannot be fitted into memory (controlled by
241 Output the final alignment to the standard output.
244 Sort by read names rather than by chromosomal coordinates
247 Approximately the maximum required memory. [500000000]
252 samtools merge [-nur] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]
254 Merge multiple sorted alignments.
255 The header reference lists of all the input BAM files, and the @SQ headers of
257 if any, must all refer to the same set of reference sequences.
258 The header reference list and (unless overridden by
264 and the headers of other files will be ignored.
272 as `@' headers to be copied to
274 replacing any header lines that would otherwise be copied from
277 is actually in SAM format, though any alignment records it may contain
281 Merge files in the specified region indicated by
285 Attach an RG tag to each alignment. The tag value is inferred from file names.
288 The input alignments are sorted by read names rather than by chromosomal
292 Uncompressed BAM output
297 samtools index <aln.bam>
299 Index sorted alignment for fast random access. Index file
305 samtools idxstats <aln.bam>
307 Retrieve and print stats in the index file. The output is TAB delimited
308 with each line consisting of reference sequence name, sequence length, #
309 mapped reads and # unmapped reads.
313 samtools faidx <ref.fasta> [region1 [...]]
315 Index reference sequence in the FASTA format or extract subsequence from
316 indexed reference sequence. If no region is specified,
318 will index the file and create
320 on the disk. If regions are speficified, the subsequences will be
321 retrieved and printed to stdout in the FASTA format. The input file can
328 samtools fixmate <in.nameSrt.bam> <out.bam>
330 Fill in mate coordinates, ISIZE and mate related flags from a
331 name-sorted alignment.
335 samtools rmdup [-sS] <input.srt.bam> <out.bam>
337 Remove potential PCR duplicates: if multiple read pairs have identical
338 external coordinates, only retain the pair with highest mapping quality.
339 In the paired-end mode, this command
341 works with FR orientation and requires ISIZE is correctly set. It does
342 not work for unpaired reads (e.g. two ends mapped to different
343 chromosomes or orphan reads).
349 Remove duplicate for single-end reads. By default, the command works for
350 paired-end reads only.
353 Treat paired-end reads and single-end reads.
358 samtools calmd [-eubSr] [-C capQcoef] <aln.bam> <ref.fasta>
360 Generate the MD tag. If the MD tag is already present, this command will
361 give a warning if the MD tag generated is different from the existing
362 tag. Output SAM by default.
368 Convert a the read base to = if it is identical to the aligned reference
369 base. Indel caller does not support the = bases at the moment.
372 Output uncompressed BAM
375 Output compressed BAM
378 The input is SAM with header lines
381 Coefficient to cap mapping quality of poorly mapped reads. See the
383 command for details. [0]
386 Perform probabilistic realignment to compute BAQ, which will be used to
392 samtools pileup [-2sSBicv] [-f in.ref.fasta] [-t in.ref_list] [-l
393 in.site_list] [-C capMapQ] [-M maxMapQ] [-T theta] [-N nHap] [-r
394 pairDiffRate] [-m mask] [-d maxIndelDepth] [-G indelPrior]
397 Print the alignment in the pileup format. In the pileup format, each
398 line represents a genomic position, consisting of chromosome name,
399 coordinate, reference base, read bases, read qualities and alignment
400 mapping qualities. Information on match, mismatch, indel, strand,
401 mapping quality and start and end of a read are all encoded at the read
402 base column. At this column, a dot stands for a match to the reference
403 base on the forward strand, a comma for a match on the reverse strand,
404 a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward
405 strand and `acgtn' for a mismatch on the reverse strand. A pattern
406 `\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this
407 reference position and the next reference position. The length of the
408 insertion is given by the integer in the pattern, followed by the
409 inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+'
410 represents a deletion from the reference. The deleted bases will be
411 presented as `*' in the following lines. Also at the read base column, a
412 symbol `^' marks the start of a read. The ASCII of the character
413 following `^' minus 33 gives the mapping quality. A symbol `$' marks the
414 end of a read segment.
418 is applied, the consensus base, Phred-scaled consensus quality, SNP
419 quality (i.e. the Phred-scaled probability of the consensus being
420 identical to the reference) and root mean square (RMS) mapping quality
421 of the reads covering the site will be inserted between the `reference
422 base' and the `read bases' columns. An indel occupies an additional
423 line. Each indel line consists of chromosome name, coordinate, a star,
424 the genotype, consensus quality, SNP quality, RMS mapping quality, #
425 covering reads, the first alllele, the second allele, # reads supporting
426 the first allele, # reads supporting the second allele and # reads
427 containing indels different from the top two alleles.
430 Since 0.1.10, the `pileup' command is deprecated by `mpileup'.
436 Disable the BAQ computation. See the
441 Call the consensus sequence. Options
442 .BR -T ", " -N ", " -I " and " -r
443 are only effective when
448 Coefficient for downgrading the mapping quality of poorly mapped
451 command for details. [0]
456 reads in the pileup for indel calling for speed up. Zero for unlimited. [1024]
459 The reference sequence in the FASTA format. Index file
465 Generate genotype likelihood in the binary GLFv3 format. This option
466 suppresses -c, -i and -s. This option is deprecated by the
471 Only output pileup lines containing indels.
474 Phred probability of an indel in sequencing/prep. [40]
477 List of sites at which pileup is output. This file is space
478 delimited. The first two columns are required to be chromosome and
479 1-based coordinate. Additional columns are ignored. It is
480 recommended to use option
483 Filter reads with flag containing bits in
488 Cap mapping quality at INT [60]
491 Number of haplotypes in the sample (>=2) [2]
494 Expected fraction of differences between a pair of haplotypes [0.001]
497 Print the mapping quality as the last column. This option makes the
498 output easier to parse, although this format is not space efficient.
501 The input file is in SAM.
504 List of reference names ane sequence lengths, in the format described
507 command. If this option is present, samtools assumes the input
509 is in SAM format; otherwise it assumes in BAM format.
513 as in the default format we may not know the mapping quality.
516 The theta parameter (error dependency coefficient) in the maq consensus
522 SAM is TAB-delimited. Apart from the header lines, which are started
523 with the `@' symbol, each alignment line consists of:
529 Col Field Description
531 1 QNAME Query (pair) NAME
533 3 RNAME Reference sequence NAME
534 4 POS 1-based leftmost POSition/coordinate of clipped sequence
535 5 MAPQ MAPping Quality (Phred-scaled)
536 6 CIAGR extended CIGAR string
537 7 MRNM Mate Reference sequence NaMe (`=' if same as RNAME)
538 8 MPOS 1-based Mate POSistion
539 9 ISIZE Inferred insert SIZE
540 10 SEQ query SEQuence on the same strand as the reference
541 11 QUAL query QUALity (ASCII-33 gives the Phred base quality)
542 12 OPT variable OPTional fields in the format TAG:VTYPE:VALUE
546 Each bit in the FLAG field is defined as:
554 0x0001 p the read is paired in sequencing
555 0x0002 P the read is mapped in a proper pair
556 0x0004 u the query sequence itself is unmapped
557 0x0008 U the mate is unmapped
558 0x0010 r strand of the query (1 for reverse)
559 0x0020 R strand of the mate
560 0x0040 1 the read is the first read in a pair
561 0x0080 2 the read is the second read in a pair
562 0x0100 s the alignment is not primary
563 0x0200 f the read fails platform/vendor quality checks
564 0x0400 d the read is either a PCR or an optical duplicate
569 Import SAM to BAM when
571 lines are present in the header:
573 samtools view -bS aln.sam > aln.bam
579 samtools faidx ref.fa
580 samtools view -bt ref.fa.fai aln.sam > aln.bam
584 is generated automatically by the
591 tag while merging sorted alignments:
593 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
594 samtools merge -rh rg.txt merged.bam ga.bam 454.bam
598 tag is determined by the file name the read is coming from. In this
611 Call SNPs and short indels for one diploid individual:
613 samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
614 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 > var.flt.vcf
618 option of varFilter controls the maximum read depth, which should be
619 adjusted to about twice the average read depth. One may consider to add
623 if mapping quality is overestimated for reads containing excessive
624 mismatches. Applying this option usually helps
626 but may not other mappers.
629 Call SNPs and short indels for multiple diploid individuals:
631 samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf
632 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf
634 Individuals are identified from the
638 header lines. Individuals can be pooled in one alignment file; one
639 individual can also be separated into multiple files. The
641 option specifies that indel candidates should be collected only from
646 Collecting indel candidates from reads sequenced by an indel-prone
647 technology may affect the performance of indel calling.
650 Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals:
652 samtools mpileup -Igf ref.fa *.bam > all.bcf
653 bcftools view -bl sites.list all.bcf > sites.bcf
654 bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
655 bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
656 bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
661 contains the list of sites with each line consisting of the reference
662 sequence name and position. The following
664 commands estimate AFS by EM.
667 Dump BAQ applied alignment for other SNP callers:
669 samtools calmd -br aln.bam > aln.baq.bam
671 It adds and corrects the
675 tags at the same time. The
677 command also comes with the
679 option, the same as the one in
688 Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
690 In merging, the input files are required to have the same number of
691 reference sequences. The requirement can be relaxed. In addition,
692 merging does not reconstruct the header dictionaries
693 automatically. Endusers have to provide the correct header. Picard is
696 Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan
697 reads or ends mapped to different chromosomes). If this is a concern,
698 please use Picard's MarkDuplicate which correctly handles these cases,
699 although a little slower.
703 Heng Li from the Sanger Institute wrote the C version of samtools. Bob
704 Handsaker from the Broad Institute implemented the BGZF library and Jue
705 Ruan from Beijing Genomics Institute wrote the RAZF library. John
706 Marshall and Petr Danecek contribute to the source code and various
707 people from the 1000 Genomes Project have contributed to the SAM format
712 Samtools website: <http://samtools.sourceforge.net>
projects / samtools.git / blob