1 .TH samtools 1 "27 October 2010" "samtools-0.1.9" "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 -agf 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 pileup [-2sSBicv] [-f in.ref.fasta] [-t in.ref_list] [-l
141 in.site_list] [-C capMapQ] [-M maxMapQ] [-T theta] [-N nHap] [-r
142 pairDiffRate] [-m mask] [-d maxIndelDepth] [-G indelPrior]
145 Print the alignment in the pileup format. In the pileup format, each
146 line represents a genomic position, consisting of chromosome name,
147 coordinate, reference base, read bases, read qualities and alignment
148 mapping qualities. Information on match, mismatch, indel, strand,
149 mapping quality and start and end of a read are all encoded at the read
150 base column. At this column, a dot stands for a match to the reference
151 base on the forward strand, a comma for a match on the reverse strand,
152 a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward
153 strand and `acgtn' for a mismatch on the reverse strand. A pattern
154 `\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this
155 reference position and the next reference position. The length of the
156 insertion is given by the integer in the pattern, followed by the
157 inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+'
158 represents a deletion from the reference. The deleted bases will be
159 presented as `*' in the following lines. Also at the read base column, a
160 symbol `^' marks the start of a read. The ASCII of the character
161 following `^' minus 33 gives the mapping quality. A symbol `$' marks the
162 end of a read segment.
166 is applied, the consensus base, Phred-scaled consensus quality, SNP
167 quality (i.e. the Phred-scaled probability of the consensus being
168 identical to the reference) and root mean square (RMS) mapping quality
169 of the reads covering the site will be inserted between the `reference
170 base' and the `read bases' columns. An indel occupies an additional
171 line. Each indel line consists of chromosome name, coordinate, a star,
172 the genotype, consensus quality, SNP quality, RMS mapping quality, #
173 covering reads, the first alllele, the second allele, # reads supporting
174 the first allele, # reads supporting the second allele and # reads
175 containing indels different from the top two alleles.
177 The position of indels is offset by -1.
183 Disable the BAQ computation. See the
188 Call the consensus sequence using SOAPsnp consensus model. Options
189 .BR -T ", " -N ", " -I " and " -r
190 are only effective when
195 Coefficient for downgrading the mapping quality of poorly mapped
198 command for details. [0]
203 reads in the pileup for indel calling for speed up. Zero for unlimited. [1024]
206 The reference sequence in the FASTA format. Index file
212 Generate genotype likelihood in the binary GLFv3 format. This option
213 suppresses -c, -i and -s. This option is deprecated by the
218 Only output pileup lines containing indels.
221 Phred probability of an indel in sequencing/prep. [40]
224 List of sites at which pileup is output. This file is space
225 delimited. The first two columns are required to be chromosome and
226 1-based coordinate. Additional columns are ignored. It is
227 recommended to use option
230 Filter reads with flag containing bits in
235 Cap mapping quality at INT [60]
238 Number of haplotypes in the sample (>=2) [2]
241 Expected fraction of differences between a pair of haplotypes [0.001]
244 Print the mapping quality as the last column. This option makes the
245 output easier to parse, although this format is not space efficient.
248 The input file is in SAM.
251 List of reference names ane sequence lengths, in the format described
254 command. If this option is present, samtools assumes the input
256 is in SAM format; otherwise it assumes in BAM format.
260 as in the default format we may not know the mapping quality.
263 The theta parameter (error dependency coefficient) in the maq consensus
269 samtools mpileup [-Bug] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-M capMapQ] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]
271 Generate BCF or pileup for one or multiple BAM files. Alignment records
272 are grouped by sample identifiers in @RG header lines. If sample
273 identifiers are absent, each input file is regarded as one sample.
279 Disable probabilistic realignment for the computation of base alignment
280 quality (BAQ). BAQ is the Phred-scaled probability of a read base being
281 misaligned. Applying this option greatly helps to reduce false SNPs
282 caused by misalignments.
285 Coefficient for downgrading mapping quality for reads containing
286 excessive mismatches. Given a read with a phred-scaled probability q of
287 being generated from the mapped position, the new mapping quality is
288 about sqrt((INT-q)/INT)*INT. A zero value disables this
289 functionality; if enabled, the recommended value is 50. [0]
292 The reference file [null]
295 Compute genotype likelihoods and output them in the binary call format (BCF).
300 except that the output is uncompressed BCF, which is preferred for pipeing.
303 File containing a list of sites where pileup or BCF is outputted [null]
306 Minimum mapping quality for an alignment to be used [0]
309 Minimum base quality for a base to be considered [13]
312 Only generate pileup in region
319 samtools reheader <in.header.sam> <in.bam>
321 Replace the header in
325 This command is much faster than replacing the header with a
326 BAM->SAM->BAM conversion.
330 samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>
332 Sort alignments by leftmost coordinates. File
334 will be created. This command may also create temporary files
335 .I <out.prefix>.%d.bam
336 when the whole alignment cannot be fitted into memory (controlled by
343 Output the final alignment to the standard output.
346 Sort by read names rather than by chromosomal coordinates
349 Approximately the maximum required memory. [500000000]
354 samtools merge [-nur] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]
356 Merge multiple sorted alignments.
357 The header reference lists of all the input BAM files, and the @SQ headers of
359 if any, must all refer to the same set of reference sequences.
360 The header reference list and (unless overridden by
366 and the headers of other files will be ignored.
374 as `@' headers to be copied to
376 replacing any header lines that would otherwise be copied from
379 is actually in SAM format, though any alignment records it may contain
383 Merge files in the specified region indicated by
387 Attach an RG tag to each alignment. The tag value is inferred from file names.
390 The input alignments are sorted by read names rather than by chromosomal
394 Uncompressed BAM output
399 samtools index <aln.bam>
401 Index sorted alignment for fast random access. Index file
407 samtools idxstats <aln.bam>
409 Retrieve and print stats in the index file. The output is TAB delimited
410 with each line consisting of reference sequence name, sequence length, #
411 mapped reads and # unmapped reads.
415 samtools faidx <ref.fasta> [region1 [...]]
417 Index reference sequence in the FASTA format or extract subsequence from
418 indexed reference sequence. If no region is specified,
420 will index the file and create
422 on the disk. If regions are speficified, the subsequences will be
423 retrieved and printed to stdout in the FASTA format. The input file can
430 samtools fixmate <in.nameSrt.bam> <out.bam>
432 Fill in mate coordinates, ISIZE and mate related flags from a
433 name-sorted alignment.
437 samtools rmdup [-sS] <input.srt.bam> <out.bam>
439 Remove potential PCR duplicates: if multiple read pairs have identical
440 external coordinates, only retain the pair with highest mapping quality.
441 In the paired-end mode, this command
443 works with FR orientation and requires ISIZE is correctly set. It does
444 not work for unpaired reads (e.g. two ends mapped to different
445 chromosomes or orphan reads).
451 Remove duplicate for single-end reads. By default, the command works for
452 paired-end reads only.
455 Treat paired-end reads and single-end reads.
460 samtools calmd [-eubSr] [-C capQcoef] <aln.bam> <ref.fasta>
462 Generate the MD tag. If the MD tag is already present, this command will
463 give a warning if the MD tag generated is different from the existing
464 tag. Output SAM by default.
470 Convert a the read base to = if it is identical to the aligned reference
471 base. Indel caller does not support the = bases at the moment.
474 Output uncompressed BAM
477 Output compressed BAM
480 The input is SAM with header lines
483 Coefficient to cap mapping quality of poorly mapped reads. See the
485 command for details. [0]
488 Perform probabilistic realignment to compute BAQ, which will be used to
494 SAM is TAB-delimited. Apart from the header lines, which are started
495 with the `@' symbol, each alignment line consists of:
501 Col Field Description
503 1 QNAME Query (pair) NAME
505 3 RNAME Reference sequence NAME
506 4 POS 1-based leftmost POSition/coordinate of clipped sequence
507 5 MAPQ MAPping Quality (Phred-scaled)
508 6 CIAGR extended CIGAR string
509 7 MRNM Mate Reference sequence NaMe (`=' if same as RNAME)
510 8 MPOS 1-based Mate POSistion
511 9 ISIZE Inferred insert SIZE
512 10 SEQ query SEQuence on the same strand as the reference
513 11 QUAL query QUALity (ASCII-33 gives the Phred base quality)
514 12 OPT variable OPTional fields in the format TAG:VTYPE:VALUE
518 Each bit in the FLAG field is defined as:
526 0x0001 p the read is paired in sequencing
527 0x0002 P the read is mapped in a proper pair
528 0x0004 u the query sequence itself is unmapped
529 0x0008 U the mate is unmapped
530 0x0010 r strand of the query (1 for reverse)
531 0x0020 R strand of the mate
532 0x0040 1 the read is the first read in a pair
533 0x0080 2 the read is the second read in a pair
534 0x0100 s the alignment is not primary
535 0x0200 f the read fails platform/vendor quality checks
536 0x0400 d the read is either a PCR or an optical duplicate
541 Import SAM to BAM when
543 lines are present in the header:
545 samtools view -bS aln.sam > aln.bam
551 samtools faidx ref.fa
552 samtools view -bt ref.fa.fai aln.sam > aln.bam
556 is generated automatically by the
563 tag while merging sorted alignments:
565 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
566 samtools merge -rh rg.txt merged.bam ga.bam 454.bam
570 tag is determined by the file name the read is coming from. In this
583 Call SNPs and short indels for one diploid individual:
585 samtools pileup -vcf ref.fa aln.bam > var.raw.plp
586 samtools.pl varFilter -D 100 var.raw.plp > var.flt.plp
587 awk '($3=="*"&&$6>=50)||($3!="*"&&$6>=20)' var.flt.plp > var.final.plp
591 option of varFilter controls the maximum read depth, which should be
592 adjusted to about twice the average read depth. One may consider to add
596 if mapping quality is overestimated for reads containing excessive
597 mismatches. Applying this option usually helps
599 but may not other mappers. It also potentially increases reference
603 Call SNPs (not short indels) for multiple diploid individuals:
605 samtools mpileup -augf ref.fa *.bam | bcftools view -bcv - > snp.raw.bcf
606 bcftools view snp.raw.bcf | vcfutils.pl filter4vcf -D 2000 | bgzip > snp.flt.vcf.gz
608 Individuals are identified from the
612 header lines. Individuals can be pooled in one alignment file; one
613 individual can also be separated into multiple files. In addition, one
614 may consider to apply
619 SNP calling with mpileup also works for single sample and has the
620 advantage of enabling more powerful filtering. The drawback is the lack
621 of short indel calling, which may be implemented in future.
624 Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals:
626 samtools mpileup -gf ref.fa *.bam > all.bcf
627 bcftools view -bl sites.list all.bcf > sites.bcf
628 bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
629 bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
630 bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
635 contains the list of sites with each line consisting of the reference
636 sequence name and position. The following
638 commands estimate AFS by EM.
641 Dump BAQ applied alignment for other SNP callers:
643 samtools calmd -br aln.bam > aln.baq.bam
645 It adds and corrects the
649 tags at the same time. The
651 command also comes with the
653 option, the same as the one in
662 Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
664 In merging, the input files are required to have the same number of
665 reference sequences. The requirement can be relaxed. In addition,
666 merging does not reconstruct the header dictionaries
667 automatically. Endusers have to provide the correct header. Picard is
670 Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan
671 reads or ends mapped to different chromosomes). If this is a concern,
672 please use Picard's MarkDuplicate which correctly handles these cases,
673 although a little slower.
677 Heng Li from the Sanger Institute wrote the C version of samtools. Bob
678 Handsaker from the Broad Institute implemented the BGZF library and Jue
679 Ruan from Beijing Genomics Institute wrote the RAZF library. John
680 Marshall and Petr Danecek contribute to the source code and various
681 people from the 1000 Genomes Project have contributed to the SAM format
686 Samtools website: <http://samtools.sourceforge.net>
projects / samtools.git / blob