1samtools(1) Bioinformatics tools samtools(1)
2
6 samtools - Utilities for the Sequence Alignment/Map (SAM) format
7 bcftools - Utilities for the Binary Call Format (BCF) and VCF
9
11 samtools view -bt ref_list.txt -o aln.bam aln.sam.gz
12 samtools sort aln.bam aln.sorted
14 samtools index aln.sorted.bam
16 samtools idxstats aln.sorted.bam
18 samtools view aln.sorted.bam chr2:20,100,000-20,200,000
20 samtools merge out.bam in1.bam in2.bam in3.bam
22 samtools faidx ref.fasta
24 samtools pileup -vcf ref.fasta aln.sorted.bam
26 samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
28 samtools tview aln.sorted.bam ref.fasta
30 bcftools index in.bcf
32 bcftools view in.bcf chr2:100-200 > out.vcf
34 bcftools view -vc in.bcf > out.vcf 2> out.afs
36
39 Samtools is a set of utilities that manipulate alignments in the BAM
40 format. It imports from and exports to the SAM (Sequence Alignment/Map)
41 format, does sorting, merging and indexing, and allows to retrieve
42 reads in any regions swiftly.
43 Samtools is designed to work on a stream. It regards an input file `-'
45 as the standard input (stdin) and an output file `-' as the standard
46 output (stdout). Several commands can thus be combined with Unix pipes.
47 Samtools always output warning and error messages to the standard error
48 output (stderr).
49 Samtools is also able to open a BAM (not SAM) file on a remote FTP or
51 HTTP server if the BAM file name starts with `ftp://' or `http://'.
52 Samtools checks the current working directory for the index file and
53 will download the index upon absence. Samtools does not retrieve the
54 entire alignment file unless it is asked to do so.
55
58 view samtools view [-bchuHS] [-t in.refList] [-o output] [-f
59 reqFlag] [-F skipFlag] [-q minMapQ] [-l library] [-r read‐
60 Group] [-R rgFile] <in.bam>|<in.sam> [region1 [...]]
61 Extract/print all or sub alignments in SAM or BAM format. If
63 no region is specified, all the alignments will be printed;
64 otherwise only alignments overlapping the specified regions
65 will be output. An alignment may be given multiple times if
66 it is overlapping several regions. A region can be presented,
67 for example, in the following format: `chr2' (the whole
68 chr2), `chr2:1000000' (region starting from 1,000,000bp) or
69 `chr2:1,000,000-2,000,000' (region between 1,000,000 and
70 2,000,000bp including the end points). The coordinate is
71 1-based.
72 OPTIONS:
74 -b Output in the BAM format.
76 -f INT Only output alignments with all bits in INT present
78 in the FLAG field. INT can be in hex in the format of
79 /^0x[0-9A-F]+/ [0]
80 -F INT Skip alignments with bits present in INT [0]
82 -h Include the header in the output.
84 -H Output the header only.
86 -l STR Only output reads in library STR [null]
88 -o FILE Output file [stdout]
90 -q INT Skip alignments with MAPQ smaller than INT [0]
92 -r STR Only output reads in read group STR [null]
94 -R FILE Output reads in read groups listed in FILE [null]
96 -S Input is in SAM. If @SQ header lines are absent, the
98 `-t' option is required.
99 -c Instead of printing the alignments, only count them
101 and print the total number. All filter options, such
102 as `-f', `-F' and `-q' , are taken into account.
103 -t FILE This file is TAB-delimited. Each line must contain
105 the reference name and the length of the reference,
106 one line for each distinct reference; additional
107 fields are ignored. This file also defines the order
108 of the reference sequences in sorting. If you run
109 `samtools faidx <ref.fa>', the resultant index file
110 <ref.fa>.fai can be used as this <in.ref_list> file.
111 -u Output uncompressed BAM. This option saves time spent
113 on compression/decomprssion and is thus preferred
114 when the output is piped to another samtools command.
115 tview samtools tview <in.sorted.bam> [ref.fasta]
118 Text alignment viewer (based on the ncurses library). In the
120 viewer, press `?' for help and press `g' to check the align‐
121 ment start from a region in the format like
122 `chr10:10,000,000' or `=10,000,000' when viewing the same
123 reference sequence.
124 mpileup samtools mpileup [-EBug] [-C capQcoef] [-r reg] [-f in.fa]
127 [-l list] [-M capMapQ] [-Q minBaseQ] [-q minMapQ] in.bam
128 [in2.bam [...]]
129 Generate BCF or pileup for one or multiple BAM files. Align‐
131 ment records are grouped by sample identifiers in @RG header
132 lines. If sample identifiers are absent, each input file is
133 regarded as one sample.
134 In the pileup format (without -uor-g), each line represents a
136 genomic position, consisting of chromosome name, coordinate,
137 reference base, read bases, read qualities and alignment map‐
138 ping qualities. Information on match, mismatch, indel,
139 strand, mapping quality and start and end of a read are all
140 encoded at the read base column. At this column, a dot stands
141 for a match to the reference base on the forward strand, a
142 comma for a match on the reverse strand, a '>' or '<' for a
143 reference skip, `ACGTN' for a mismatch on the forward strand
144 and `acgtn' for a mismatch on the reverse strand. A pattern
145 `\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion
146 between this reference position and the next reference posi‐
147 tion. The length of the insertion is given by the integer in
148 the pattern, followed by the inserted sequence. Similarly, a
149 pattern `-[0-9]+[ACGTNacgtn]+' represents a deletion from the
150 reference. The deleted bases will be presented as `*' in the
151 following lines. Also at the read base column, a symbol `^'
152 marks the start of a read. The ASCII of the character follow‐
153 ing `^' minus 33 gives the mapping quality. A symbol `$'
154 marks the end of a read segment.
155 Input Options:
157 -6 Assume the quality is in the Illumina 1.3+ encod‐
159 ing. -A Do not skip anomalous read pairs in vari‐
160 ant calling.
161 -B Disable probabilistic realignment for the computa‐
163 tion of base alignment quality (BAQ). BAQ is the
164 Phred-scaled probability of a read base being mis‐
165 aligned. Applying this option greatly helps to
166 reduce false SNPs caused by misalignments.
167 -b FILE List of input BAM files, one file per line [null]
169 -C INT Coefficient for downgrading mapping quality for
171 reads containing excessive mismatches. Given a read
172 with a phred-scaled probability q of being gener‐
173 ated from the mapped position, the new mapping
174 quality is about sqrt((INT-q)/INT)*INT. A zero
175 value disables this functionality; if enabled, the
176 recommended value for BWA is 50. [0]
177 -d INT At a position, read maximally INT reads per input
179 BAM. [250]
180 -E Extended BAQ computation. This option helps sensi‐
182 tivity especially for MNPs, but may hurt speci‐
183 ficity a little bit.
184 -f FILE The faidx-indexed reference file in the FASTA for‐
186 mat. The file can be optionally compressed by
187 razip. [null]
188 -l FILE BED or position list file containing a list of
190 regions or sites where pileup or BCF should be gen‐
191 erated [null]
192 -q INT Minimum mapping quality for an alignment to be used
194 [0]
195 -Q INT Minimum base quality for a base to be considered
197 [13]
198 -r STR Only generate pileup in region STR [all sites]
200 Output Options:
202 -D Output per-sample read depth
205 -g Compute genotype likelihoods and output them in the
207 binary call format (BCF).
208 -S Output per-sample Phred-scaled strand bias P-value
210 -u Similar to -g except that the output is uncom‐
212 pressed BCF, which is preferred for piping.
213 Options for Genotype Likelihood Computation (for -g or -u):
216 -e INT Phred-scaled gap extension sequencing error proba‐
219 bility. Reducing INT leads to longer indels. [20]
220 -h INT Coefficient for modeling homopolymer errors. Given
222 an l-long homopolymer run, the sequencing error of
223 an indel of size s is modeled as INT*s/l. [100]
224 -I Do not perform INDEL calling
226 -L INT Skip INDEL calling if the average per-sample depth
228 is above INT. [250]
229 -o INT Phred-scaled gap open sequencing error probability.
231 Reducing INT leads to more indel calls. [40]
232 -P STR Comma dilimited list of platforms (determined by
234 @RG-PL) from which indel candidates are obtained.
235 It is recommended to collect indel candidates from
236 sequencing technologies that have low indel error
237 rate such as ILLUMINA. [all]
238 reheader samtools reheader <in.header.sam> <in.bam>
241 Replace the header in in.bam with the header in
243 in.header.sam. This command is much faster than replacing
244 the header with a BAM->SAM->BAM conversion.
245 cat samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam>
248 [ ... ]
249 Concatenate BAMs. The sequence dictionary of each input BAM
251 must be identical, although this command does not check this.
252 This command uses a similar trick to reheader which enables
253 fast BAM concatenation.
254 sort samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>
257 Sort alignments by leftmost coordinates. File <out.pre‐
259 fix>.bam will be created. This command may also create tempo‐
260 rary files <out.prefix>.%d.bam when the whole alignment can‐
261 not be fitted into memory (controlled by option -m).
262 OPTIONS:
264 -o Output the final alignment to the standard output.
266 -n Sort by read names rather than by chromosomal coordi‐
268 nates
269 -m INT Approximately the maximum required memory.
271 [500000000]
272 merge samtools merge [-nur1f] [-h inh.sam] [-R reg] <out.bam>
275 <in1.bam> <in2.bam> [...]
276 Merge multiple sorted alignments. The header reference lists
278 of all the input BAM files, and the @SQ headers of inh.sam,
279 if any, must all refer to the same set of reference
280 sequences. The header reference list and (unless overridden
281 by -h) `@' headers of in1.bam will be copied to out.bam, and
282 the headers of other files will be ignored.
283 OPTIONS:
285 -1 Use zlib compression level 1 to comrpess the output
287 -f Force to overwrite the output file if present.
289 -h FILE Use the lines of FILE as `@' headers to be copied to
291 out.bam, replacing any header lines that would other‐
292 wise be copied from in1.bam. (FILE is actually in
293 SAM format, though any alignment records it may con‐
294 tain are ignored.)
295 -n The input alignments are sorted by read names rather
297 than by chromosomal coordinates
298 -R STR Merge files in the specified region indicated by STR
300 [null]
301 -r Attach an RG tag to each alignment. The tag value is
303 inferred from file names.
304 -u Uncompressed BAM output
306 index samtools index <aln.bam>
309 Index sorted alignment for fast random access. Index file
311 <aln.bam>.bai will be created.
312 idxstats samtools idxstats <aln.bam>
315 Retrieve and print stats in the index file. The output is TAB
317 delimited with each line consisting of reference sequence
318 name, sequence length, # mapped reads and # unmapped reads.
319 faidx samtools faidx <ref.fasta> [region1 [...]]
322 Index reference sequence in the FASTA format or extract sub‐
324 sequence from indexed reference sequence. If no region is
325 specified, faidx will index the file and create
326 <ref.fasta>.fai on the disk. If regions are speficified, the
327 subsequences will be retrieved and printed to stdout in the
328 FASTA format. The input file can be compressed in the RAZF
329 format.
330 fixmate samtools fixmate <in.nameSrt.bam> <out.bam>
333 Fill in mate coordinates, ISIZE and mate related flags from a
335 name-sorted alignment.
336 rmdup samtools rmdup [-sS] <input.srt.bam> <out.bam>
339 Remove potential PCR duplicates: if multiple read pairs have
341 identical external coordinates, only retain the pair with
342 highest mapping quality. In the paired-end mode, this com‐
343 mand ONLY works with FR orientation and requires ISIZE is
344 correctly set. It does not work for unpaired reads (e.g. two
345 ends mapped to different chromosomes or orphan reads).
346 OPTIONS:
348 -s Remove duplicate for single-end reads. By default,
350 the command works for paired-end reads only.
351 -S Treat paired-end reads and single-end reads.
353 calmd samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta>
356 Generate the MD tag. If the MD tag is already present, this
358 command will give a warning if the MD tag generated is dif‐
359 ferent from the existing tag. Output SAM by default.
360 OPTIONS:
362 -A When used jointly with -r this option overwrites the
364 original base quality.
365 -e Convert a the read base to = if it is identical to
367 the aligned reference base. Indel caller does not
368 support the = bases at the moment.
369 -u Output uncompressed BAM
371 -b Output compressed BAM
373 -S The input is SAM with header lines
375 -C INT Coefficient to cap mapping quality of poorly mapped
377 reads. See the pileup command for details. [0]
378 -r Compute the BQ tag (without -A) or cap base quality
380 by BAQ (with -A).
381 -E Extended BAQ calculation. This option trades speci‐
383 ficity for sensitivity, though the effect is minor.
384 targetcut samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1
387 em1] [-2 em2] [-f ref] <in.bam>
388 This command identifies target regions by examining the con‐
390 tinuity of read depth, computes haploid consensus sequences
391 of targets and outputs a SAM with each sequence corresponding
392 to a target. When option -f is in use, BAQ will be applied.
393 This command is only designed for cutting fosmid clones from
394 fosmid pool sequencing [Ref. Kitzman et al. (2010)].
395 phase samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q
398 minBaseQ] <in.bam>
399 Call and phase heterozygous SNPs. OPTIONS:
401 -A Drop reads with ambiguous phase.
403 -b STR Prefix of BAM output. When this option is in use,
405 phase-0 reads will be saved in file STR.0.bam and
406 phase-1 reads in STR.1.bam. Phase unknown reads will
407 be randomly allocated to one of the two files.
408 Chimeric reads with switch errors will be saved in
409 STR.chimeric.bam. [null]
410 -F Do not attempt to fix chimeric reads.
412 -k INT Maximum length for local phasing. [13]
414 -q INT Minimum Phred-scaled LOD to call a heterozygote. [40]
416 -Q INT Minimum base quality to be used in het calling. [13]
418
421 view bcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s
422 listSample] [-i gapSNPratio] [-t mutRate] [-p varThres] [-P
423 prior] [-1 nGroup1] [-d minFrac] [-U nPerm] [-X permThres]
424 [-T trioType] in.bcf [region]
425 Convert between BCF and VCF, call variant candidates and
427 estimate allele frequencies.
428 Input/Output Options:
431 -A Retain all possible alternate alleles at variant
433 sites. By default, the view command discards
434 unlikely alleles.
435 -b Output in the BCF format. The default is VCF.
437 -D FILE Sequence dictionary (list of chromosome names) for
439 VCF->BCF conversion [null]
440 -F Indicate PL is generated by r921 or before (order‐
442 ing is different).
443 -G Suppress all individual genotype information.
445 -l FILE List of sites at which information are outputted
447 [all sites]
448 -N Skip sites where the REF field is not A/C/G/T
450 -Q Output the QCALL likelihood format
452 -s FILE List of samples to use. The first column in the
454 input gives the sample names and the second gives
455 the ploidy, which can only be 1 or 2. When the 2nd
456 column is absent, the sample ploidy is assumed to
457 be 2. In the output, the ordering of samples will
458 be identical to the one in FILE. [null]
459 -S The input is VCF instead of BCF.
461 -u Uncompressed BCF output (force -b).
463 Consensus/Variant Calling Options:
465 -c Call variants using Bayesian inference. This option
467 automatically invokes option -e.
468 -d FLOAT When -v is in use, skip loci where the fraction of
470 samples covered by reads is below FLOAT. [0]
471 -e Perform max-likelihood inference only, including
473 estimating the site allele frequency, testing
474 Hardy-Weinberg equlibrium and testing associations
475 with LRT.
476 -g Call per-sample genotypes at variant sites (force
478 -c)
479 -i FLOAT Ratio of INDEL-to-SNP mutation rate [0.15]
481 -p FLOAT A site is considered to be a variant if
483 P(ref|D)<FLOAT [0.5]
484 -P STR Prior or initial allele frequency spectrum. If STR
486 can be full, cond2, flat or the file consisting of
487 error output from a previous variant calling run.
488 -t FLOAT Scaled muttion rate for variant calling [0.001]
490 -T STR Enable pair/trio calling. For trio calling, option
492 -s is usually needed to be applied to configure the
493 trio members and their ordering. In the file sup‐
494 plied to the option -s, the first sample must be
495 the child, the second the father and the third the
496 mother. The valid values of STR are `pair',
497 `trioauto', `trioxd' and `trioxs', where `pair'
498 calls differences between two input samples, and
499 `trioxd' (`trioxs') specifies that the input is
500 from the X chromosome non-PAR regions and the child
501 is a female (male). [null]
502 -v Output variant sites only (force -c)
504 Contrast Calling and Association Test Options:
506 -1 INT Number of group-1 samples. This option is used for
508 dividing the samples into two groups for contrast
509 SNP calling or association test. When this option
510 is in use, the following VCF INFO will be out‐
511 putted: PC2, PCHI2 and QCHI2. [0]
512 -U INT Number of permutations for association test (effec‐
514 tive only with -1) [0]
515 -X FLOAT Only perform permutations for P(chi^2)<FLOAT
517 (effective only with -U) [0.01]
518 index bcftools index in.bcf
521 Index sorted BCF for random access.
523 cat bcftools cat in1.bcf [in2.bcf [...]]]
526 Concatenate BCF files. The input files are required to be
528 sorted and have identical samples appearing in the same
529 order.
530
532 Sequence Alignment/Map (SAM) format is TAB-delimited. Apart from the
533 header lines, which are started with the `@' symbol, each alignment
534 line consists of:
535 ┌────┬───────┬──────────────────────────────────────────────────────────┐
538 │Col │ Field │ Description │
539 ├────┼───────┼──────────────────────────────────────────────────────────┤
540 │ 1 │ QNAME │ Query template/pair NAME │
541 │ 2 │ FLAG │ bitwise FLAG │
542 │ 3 │ RNAME │ Reference sequence NAME │
543 │ 4 │ POS │ 1-based leftmost POSition/coordinate of clipped sequence │
544 │ 5 │ MAPQ │ MAPping Quality (Phred-scaled) │
545 │ 6 │ CIAGR │ extended CIGAR string │
546 │ 7 │ MRNM │ Mate Reference sequence NaMe (`=' if same as RNAME) │
547 │ 8 │ MPOS │ 1-based Mate POSistion │
548 │ 9 │ TLEN │ inferred Template LENgth (insert size) │
549 │10 │ SEQ │ query SEQuence on the same strand as the reference │
550 │11 │ QUAL │ query QUALity (ASCII-33 gives the Phred base quality) │
551 │12+ │ OPT │ variable OPTional fields in the format TAG:VTYPE:VALUE │
552 └────┴───────┴──────────────────────────────────────────────────────────┘
553 Each bit in the FLAG field is defined as:
555 ┌───────┬─────┬──────────────────────────────────────────────────┐
558 │ Flag │ Chr │ Description │
559 ├───────┼─────┼──────────────────────────────────────────────────┤
560 │0x0001 │ p │ the read is paired in sequencing │
561 │0x0002 │ P │ the read is mapped in a proper pair │
562 │0x0004 │ u │ the query sequence itself is unmapped │
563 │0x0008 │ U │ the mate is unmapped │
564 │0x0010 │ r │ strand of the query (1 for reverse) │
565 │0x0020 │ R │ strand of the mate │
566 │0x0040 │ 1 │ the read is the first read in a pair │
567 │0x0080 │ 2 │ the read is the second read in a pair │
568 │0x0100 │ s │ the alignment is not primary │
569 │0x0200 │ f │ the read fails platform/vendor quality checks │
570 │0x0400 │ d │ the read is either a PCR or an optical duplicate │
571 └───────┴─────┴──────────────────────────────────────────────────┘
572 where the second column gives the string representation of the FLAG
573 field.
574
577 The Variant Call Format (VCF) is a TAB-delimited format with each data
578 line consists of the following fields:
579 ┌────┬────────┬──────────────────────────────────────────────────────────────┐
581 │Col │ Field │ Description │
582 ├────┼────────┼──────────────────────────────────────────────────────────────┤
583 │ 1 │ CHROM │ CHROMosome name │
584 │ 2 │ POS │ the left-most POSition of the variant │
585 │ 3 │ ID │ unique variant IDentifier │
586 │ 4 │ REF │ the REFerence allele │
587 │ 5 │ ALT │ the ALTernate allele(s), separated by comma │
588 │ 6 │ QUAL │ variant/reference QUALity │
589 │ 7 │ FILTER │ FILTers applied │
590 │ 8 │ INFO │ INFOrmation related to the variant, separated by semi-colon │
591 │ 9 │ FORMAT │ FORMAT of the genotype fields, separated by colon (optional) │
592 │10+ │ SAMPLE │ SAMPLE genotypes and per-sample information (optional) │
593 └────┴────────┴──────────────────────────────────────────────────────────────┘
594 The following table gives the INFO tags used by samtools and bcftools.
596┌──────┬───────────┬──────────────────────────────────────────────────────────────────────────────────────┐
599│ Tag │ Format │ Description │
600├──────┼───────────┼──────────────────────────────────────────────────────────────────────────────────────┤
601│AF1 │ double │ Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele │
602│DP │ int │ Raw read depth (without quality filtering) │
603│DP4 │ int[4] │ # high-quality reference forward bases, ref reverse, alternate for and alt rev bases │
604│FQ │ int │ Consensus quality. Positive: sample genotypes different; negative: otherwise │
605│MQ │ int │ Root-Mean-Square mapping quality of covering reads │
606│PC2 │ int[2] │ Phred probability of AF in group1 samples being larger (,smaller) than in group2 │
607│PCHI2 │ double │ Posterior weighted chi^2 P-value between group1 and group2 samples │
608│PV4 │ double[4] │ P-value for strand bias, baseQ bias, mapQ bias and tail distance bias │
609│QCHI2 │ int │ Phred-scaled PCHI2 │
610│RP │ int │ # permutations yielding a smaller PCHI2 │
611│CLR │ int │ Phred log ratio of genotype likelihoods with and without the trio/pair constraint │
612│UGT │ string │ Most probable genotype configuration without the trio constraint │
613│CGT │ string │ Most probable configuration with the trio constraint │
614└──────┴───────────┴──────────────────────────────────────────────────────────────────────────────────────┘
615
617 o Import SAM to BAM when @SQ lines are present in the header:
618 samtools view -bS aln.sam > aln.bam
620 If @SQ lines are absent:
622 samtools faidx ref.fa
624 samtools view -bt ref.fa.fai aln.sam > aln.bam
625 where ref.fa.fai is generated automatically by the faidx command.
627 o Attach the RG tag while merging sorted alignments:
630 perl -e 'print "@RG\tID:ga\tSM:hs\tLB:ga\tPL:Illu‐
632 mina\n@RG\tID:454\tSM:hs\tLB:454\tPL:454\n"' > rg.txt
633 samtools merge -rh rg.txt merged.bam ga.bam 454.bam
634 The value in a RG tag is determined by the file name the read is com‐
636 ing from. In this example, in the merged.bam, reads from ga.bam will
637 be attached RG:Z:ga, while reads from 454.bam will be attached
638 RG:Z:454.
639 o Call SNPs and short INDELs for one diploid individual:
642 samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - >
644 var.raw.bcf
645 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 >
646 var.flt.vcf
647 The -D option of varFilter controls the maximum read depth, which
649 should be adjusted to about twice the average read depth. One may
650 consider to add -C50 to mpileup if mapping quality is overestimated
651 for reads containing excessive mismatches. Applying this option usu‐
652 ally helps BWA-short but may not other mappers.
653 o Generate the consensus sequence for one diploid individual:
656 samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfu‐
658 tils.pl vcf2fq > cns.fq
659 o Call somatic mutations from a pair of samples:
662 samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -
664 > var.bcf
665 In the output INFO field, CLR gives the Phred-log ratio between the
667 likelihood by treating the two samples independently, and the likeli‐
668 hood by requiring the genotype to be identical. This CLR is effec‐
669 tively a score measuring the confidence of somatic calls. The higher
670 the better.
671 o Call de novo and somatic mutations from a family trio:
674 samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair
676 -s samples.txt - > var.bcf
677 File samples.txt should consist of three lines specifying the member
679 and order of samples (in the order of child-father-mother). Simi‐
680 larly, CLR gives the Phred-log likelihood ratio with and without the
681 trio constraint. UGT shows the most likely genotype configuration
682 without the trio constraint, and CGT gives the most likely genotype
683 configuration satisfying the trio constraint.
684 o Phase one individual:
687 samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - >
689 phase.out
690 The calmd command is used to reduce false heterozygotes around
692 INDELs.
693 o Call SNPs and short indels for multiple diploid individuals:
696 samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view
698 -bcvg - > var.raw.bcf
699 bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 >
700 var.flt.vcf
701 Individuals are identified from the SM tags in the @RG header lines.
703 Individuals can be pooled in one alignment file; one individual can
704 also be separated into multiple files. The -P option specifies that
705 indel candidates should be collected only from read groups with the
706 @RG-PL tag set to ILLUMINA. Collecting indel candidates from reads
707 sequenced by an indel-prone technology may affect the performance of
708 indel calling.
709 o Derive the allele frequency spectrum (AFS) on a list of sites from
712 multiple individuals:
713 samtools mpileup -Igf ref.fa *.bam > all.bcf
715 bcftools view -bl sites.list all.bcf > sites.bcf
716 bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
717 bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
718 bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
719 ......
720 where sites.list contains the list of sites with each line consisting
722 of the reference sequence name and position. The following bcftools
723 commands estimate AFS by EM.
724 o Dump BAQ applied alignment for other SNP callers:
727 samtools calmd -bAr aln.bam > aln.baq.bam
729 It adds and corrects the NM and MD tags at the same time. The calmd
731 command also comes with the -C option, the same as the one in pileup
732 and mpileup. Apply if it helps.
733
736 o Unaligned words used in bam_import.c, bam_endian.h, bam.c and
737 bam_aux.c.
738 o Samtools paired-end rmdup does not work for unpaired reads (e.g.
740 orphan reads or ends mapped to different chromosomes). If this is a
741 concern, please use Picard's MarkDuplicate which correctly handles
742 these cases, although a little slower.
743
746 Heng Li from the Sanger Institute wrote the C version of samtools. Bob
747 Handsaker from the Broad Institute implemented the BGZF library and Jue
748 Ruan from Beijing Genomics Institute wrote the RAZF library. John Mar‐
749 shall and Petr Danecek contribute to the source code and various people
750 from the 1000 Genomes Project have contributed to the SAM format speci‐
751 fication.
752
755 Samtools website: <http://samtools.sourceforge.net>
756samtools-0.1.17 05 July 2011 samtools(1)