Repository navigation
Expand file tree
/
Copy pathsamtools.1
More file actions
998 lines (902 loc) · 25.9 KB
/
Copy pathsamtools.1
File metadata and controls
998 lines (902 loc) · 25.9 KB
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
.TH samtools 1 "2 September 2011" "samtools-0.1.18" "Bioinformatics tools"
.SH NAME
.PP
samtools - Utilities for the Sequence Alignment/Map (SAM) format
bcftools - Utilities for the Binary Call Format (BCF) and VCF
.SH SYNOPSIS
.PP
samtools view -bt ref_list.txt -o aln.bam aln.sam.gz
.PP
samtools sort aln.bam aln.sorted
.PP
samtools index aln.sorted.bam
.PP
samtools idxstats aln.sorted.bam
.PP
samtools view aln.sorted.bam chr2:20,100,000-20,200,000
.PP
samtools merge out.bam in1.bam in2.bam in3.bam
.PP
samtools faidx ref.fasta
.PP
samtools pileup -vcf ref.fasta aln.sorted.bam
.PP
samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
.PP
samtools tview aln.sorted.bam ref.fasta
.PP
bcftools index in.bcf
.PP
bcftools view in.bcf chr2:100-200 > out.vcf
.PP
bcftools view -vc in.bcf > out.vcf 2> out.afs
.SH DESCRIPTION
.PP
Samtools is a set of utilities that manipulate alignments in the BAM
format. It imports from and exports to the SAM (Sequence Alignment/Map)
format, does sorting, merging and indexing, and allows to retrieve reads
in any regions swiftly.
Samtools is designed to work on a stream. It regards an input file `-'
as the standard input (stdin) and an output file `-' as the standard
output (stdout). Several commands can thus be combined with Unix
pipes. Samtools always output warning and error messages to the standard
error output (stderr).
Samtools is also able to open a BAM (not SAM) file on a remote FTP or
HTTP server if the BAM file name starts with `ftp://' or `http://'.
Samtools checks the current working directory for the index file and
will download the index upon absence. Samtools does not retrieve the
entire alignment file unless it is asked to do so.
.SH SAMTOOLS COMMANDS AND OPTIONS
.TP 10
.B view
samtools view [-bchuHS] [-t in.refList] [-o output] [-f reqFlag] [-F
skipFlag] [-q minMapQ] [-l library] [-r readGroup] [-R rgFile] <in.bam>|<in.sam> [region1 [...]]
Extract/print all or sub alignments in SAM or BAM format. If no region
is specified, all the alignments will be printed; otherwise only
alignments overlapping the specified regions will be output. An
alignment may be given multiple times if it is overlapping several
regions. A region can be presented, for example, in the following
format: `chr2' (the whole chr2), `chr2:1000000' (region starting from
1,000,000bp) or `chr2:1,000,000-2,000,000' (region between 1,000,000 and
2,000,000bp including the end points). The coordinate is 1-based.
.B OPTIONS:
.RS
.TP 10
.B -b
Output in the BAM format.
.TP
.BI -f \ INT
Only output alignments with all bits in INT present in the FLAG
field. INT can be in hex in the format of /^0x[0-9A-F]+/ [0]
.TP
.BI -F \ INT
Skip alignments with bits present in INT [0]
.TP
.B -h
Include the header in the output.
.TP
.B -H
Output the header only.
.TP
.BI -l \ STR
Only output reads in library STR [null]
.TP
.BI -o \ FILE
Output file [stdout]
.TP
.BI -q \ INT
Skip alignments with MAPQ smaller than INT [0]
.TP
.BI -r \ STR
Only output reads in read group STR [null]
.TP
.BI -R \ FILE
Output reads in read groups listed in
.I FILE
[null]
.TP
.BI -s \ FLOAT
Fraction of templates/pairs to subsample; the integer part is treated as the
seed for the random number generator [-1]
.TP
.B -S
Input is in SAM. If @SQ header lines are absent, the
.B `-t'
option is required.
.TP
.B -c
Instead of printing the alignments, only count them and print the
total number. All filter options, such as
.B `-f',
.B `-F'
and
.B `-q'
, are taken into account.
.TP
.BI -t \ FILE
This file is TAB-delimited. Each line must contain the reference name
and the length of the reference, one line for each distinct reference;
additional fields are ignored. This file also defines the order of the
reference sequences in sorting. If you run `samtools faidx <ref.fa>',
the resultant index file
.I <ref.fa>.fai
can be used as this
.I <in.ref_list>
file.
.TP
.B -u
Output uncompressed BAM. This option saves time spent on
compression/decomprssion and is thus preferred when the output is piped
to another samtools command.
.RE
.TP
.B tview
samtools tview <in.sorted.bam> [ref.fasta]
Text alignment viewer (based on the ncurses library). In the viewer,
press `?' for help and press `g' to check the alignment start from a
region in the format like `chr10:10,000,000' or `=10,000,000' when
viewing the same reference sequence.
.TP
.B mpileup
.B samtools mpileup
.RB [ \-EBug ]
.RB [ \-C
.IR capQcoef ]
.RB [ \-r
.IR reg ]
.RB [ \-f
.IR in.fa ]
.RB [ \-l
.IR list ]
.RB [ \-M
.IR capMapQ ]
.RB [ \-Q
.IR minBaseQ ]
.RB [ \-q
.IR minMapQ ]
.I in.bam
.RI [ in2.bam
.RI [ ... ]]
Generate BCF or pileup for one or multiple BAM files. Alignment records
are grouped by sample identifiers in @RG header lines. If sample
identifiers are absent, each input file is regarded as one sample.
In the pileup format (without
.BR -u or -g ),
each
line represents a genomic position, consisting of chromosome name,
coordinate, reference base, read bases, read qualities and alignment
mapping qualities. Information on match, mismatch, indel, strand,
mapping quality and start and end of a read are all encoded at the read
base column. At this column, a dot stands for a match to the reference
base on the forward strand, a comma for a match on the reverse strand,
a '>' or '<' for a reference skip, `ACGTN' for a mismatch on the forward
strand and `acgtn' for a mismatch on the reverse strand. A pattern
`\\+[0-9]+[ACGTNacgtn]+' indicates there is an insertion between this
reference position and the next reference position. The length of the
insertion is given by the integer in the pattern, followed by the
inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+'
represents a deletion from the reference. The deleted bases will be
presented as `*' in the following lines. Also at the read base column, a
symbol `^' marks the start of a read. The ASCII of the character
following `^' minus 33 gives the mapping quality. A symbol `$' marks the
end of a read segment.
.B Input Options:
.RS
.TP 10
.B -6
Assume the quality is in the Illumina 1.3+ encoding.
.B -A
Do not skip anomalous read pairs in variant calling.
.TP
.B -B
Disable probabilistic realignment for the computation of base alignment
quality (BAQ). BAQ is the Phred-scaled probability of a read base being
misaligned. Applying this option greatly helps to reduce false SNPs
caused by misalignments.
.TP
.BI -b \ FILE
List of input BAM files, one file per line [null]
.TP
.BI -C \ INT
Coefficient for downgrading mapping quality for reads containing
excessive mismatches. Given a read with a phred-scaled probability q of
being generated from the mapped position, the new mapping quality is
about sqrt((INT-q)/INT)*INT. A zero value disables this
functionality; if enabled, the recommended value for BWA is 50. [0]
.TP
.BI -d \ INT
At a position, read maximally
.I INT
reads per input BAM. [250]
.TP
.B -E
Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt
specificity a little bit.
.TP
.BI -f \ FILE
The
.BR faidx -indexed
reference file in the FASTA format. The file can be optionally compressed by
.BR razip .
[null]
.TP
.BI -l \ FILE
BED or position list file containing a list of regions or sites where pileup or BCF should be generated [null]
.TP
.BI -q \ INT
Minimum mapping quality for an alignment to be used [0]
.TP
.BI -Q \ INT
Minimum base quality for a base to be considered [13]
.TP
.BI -r \ STR
Only generate pileup in region
.I STR
[all sites]
.TP
.B Output Options:
.TP
.B -D
Output per-sample read depth
.TP
.B -g
Compute genotype likelihoods and output them in the binary call format (BCF).
.TP
.B -S
Output per-sample Phred-scaled strand bias P-value
.TP
.B -u
Similar to
.B -g
except that the output is uncompressed BCF, which is preferred for piping.
.TP
.B Options for Genotype Likelihood Computation (for -g or -u):
.TP
.BI -e \ INT
Phred-scaled gap extension sequencing error probability. Reducing
.I INT
leads to longer indels. [20]
.TP
.BI -h \ INT
Coefficient for modeling homopolymer errors. Given an
.IR l -long
homopolymer
run, the sequencing error of an indel of size
.I s
is modeled as
.IR INT * s / l .
[100]
.TP
.B -I
Do not perform INDEL calling
.TP
.BI -L \ INT
Skip INDEL calling if the average per-sample depth is above
.IR INT .
[250]
.TP
.BI -o \ INT
Phred-scaled gap open sequencing error probability. Reducing
.I INT
leads to more indel calls. [40]
.TP
.BI -P \ STR
Comma dilimited list of platforms (determined by
.BR @RG-PL )
from which indel candidates are obtained. It is recommended to collect
indel candidates from sequencing technologies that have low indel error
rate such as ILLUMINA. [all]
.RE
.TP
.B reheader
samtools reheader <in.header.sam> <in.bam>
Replace the header in
.I in.bam
with the header in
.I in.header.sam.
This command is much faster than replacing the header with a
BAM->SAM->BAM conversion.
.TP
.B cat
samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ]
Concatenate BAMs. The sequence dictionary of each input BAM must be identical,
although this command does not check this. This command uses a similar trick
to
.B reheader
which enables fast BAM concatenation.
.TP
.B sort
samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>
Sort alignments by leftmost coordinates. File
.I <out.prefix>.bam
will be created. This command may also create temporary files
.I <out.prefix>.%d.bam
when the whole alignment cannot be fitted into memory (controlled by
option -m).
.B OPTIONS:
.RS
.TP 8
.B -o
Output the final alignment to the standard output.
.TP
.B -n
Sort by read names rather than by chromosomal coordinates
.TP
.BI -m \ INT
Approximately the maximum required memory. [500000000]
.RE
.TP
.B merge
samtools merge [-nur1f] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...]
Merge multiple sorted alignments.
The header reference lists of all the input BAM files, and the @SQ headers of
.IR inh.sam ,
if any, must all refer to the same set of reference sequences.
The header reference list and (unless overridden by
.BR -h )
`@' headers of
.I in1.bam
will be copied to
.IR out.bam ,
and the headers of other files will be ignored.
.B OPTIONS:
.RS
.TP 8
.B -1
Use zlib compression level 1 to comrpess the output
.TP
.B -f
Force to overwrite the output file if present.
.TP 8
.BI -h \ FILE
Use the lines of
.I FILE
as `@' headers to be copied to
.IR out.bam ,
replacing any header lines that would otherwise be copied from
.IR in1.bam .
.RI ( FILE
is actually in SAM format, though any alignment records it may contain
are ignored.)
.TP
.B -n
The input alignments are sorted by read names rather than by chromosomal
coordinates
.TP
.BI -R \ STR
Merge files in the specified region indicated by
.I STR
[null]
.TP
.B -r
Attach an RG tag to each alignment. The tag value is inferred from file names.
.TP
.B -u
Uncompressed BAM output
.RE
.TP
.B index
samtools index <aln.bam>
Index sorted alignment for fast random access. Index file
.I <aln.bam>.bai
will be created.
.TP
.B idxstats
samtools idxstats <aln.bam>
Retrieve and print stats in the index file. The output is TAB delimited
with each line consisting of reference sequence name, sequence length, #
mapped reads and # unmapped reads.
.TP
.B faidx
samtools faidx <ref.fasta> [region1 [...]]
Index reference sequence in the FASTA format or extract subsequence from
indexed reference sequence. If no region is specified,
.B faidx
will index the file and create
.I <ref.fasta>.fai
on the disk. If regions are speficified, the subsequences will be
retrieved and printed to stdout in the FASTA format. The input file can
be compressed in the
.B RAZF
format.
.TP
.B fixmate
samtools fixmate <in.nameSrt.bam> <out.bam>
Fill in mate coordinates, ISIZE and mate related flags from a
name-sorted alignment.
.TP
.B rmdup
samtools rmdup [-sS] <input.srt.bam> <out.bam>
Remove potential PCR duplicates: if multiple read pairs have identical
external coordinates, only retain the pair with highest mapping quality.
In the paired-end mode, this command
.B ONLY
works with FR orientation and requires ISIZE is correctly set. It does
not work for unpaired reads (e.g. two ends mapped to different
chromosomes or orphan reads).
.B OPTIONS:
.RS
.TP 8
.B -s
Remove duplicate for single-end reads. By default, the command works for
paired-end reads only.
.TP 8
.B -S
Treat paired-end reads and single-end reads.
.RE
.TP
.B calmd
samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta>
Generate the MD tag. If the MD tag is already present, this command will
give a warning if the MD tag generated is different from the existing
tag. Output SAM by default.
.B OPTIONS:
.RS
.TP 8
.B -A
When used jointly with
.B -r
this option overwrites the original base quality.
.TP 8
.B -e
Convert a the read base to = if it is identical to the aligned reference
base. Indel caller does not support the = bases at the moment.
.TP
.B -u
Output uncompressed BAM
.TP
.B -b
Output compressed BAM
.TP
.B -S
The input is SAM with header lines
.TP
.BI -C \ INT
Coefficient to cap mapping quality of poorly mapped reads. See the
.B pileup
command for details. [0]
.TP
.B -r
Compute the BQ tag (without -A) or cap base quality by BAQ (with -A).
.TP
.B -E
Extended BAQ calculation. This option trades specificity for sensitivity, though the
effect is minor.
.RE
.TP
.B targetcut
samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam>
This command identifies target regions by examining the continuity of read depth, computes
haploid consensus sequences of targets and outputs a SAM with each sequence corresponding
to a target. When option
.B -f
is in use, BAQ will be applied. This command is
.B only
designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].
.RE
.TP
.B phase
samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam>
Call and phase heterozygous SNPs.
.B OPTIONS:
.RS
.TP 8
.B -A
Drop reads with ambiguous phase.
.TP 8
.BI -b \ STR
Prefix of BAM output. When this option is in use, phase-0 reads will be saved in file
.BR STR .0.bam
and phase-1 reads in
.BR STR .1.bam.
Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads
with switch errors will be saved in
.BR STR .chimeric.bam.
[null]
.TP
.B -F
Do not attempt to fix chimeric reads.
.TP
.BI -k \ INT
Maximum length for local phasing. [13]
.TP
.BI -q \ INT
Minimum Phred-scaled LOD to call a heterozygote. [40]
.TP
.BI -Q \ INT
Minimum base quality to be used in het calling. [13]
.RE
.SH BCFTOOLS COMMANDS AND OPTIONS
.TP 10
.B view
.B bcftools view
.RB [ \-AbFGNQSucgv ]
.RB [ \-D
.IR seqDict ]
.RB [ \-l
.IR listLoci ]
.RB [ \-s
.IR listSample ]
.RB [ \-i
.IR gapSNPratio ]
.RB [ \-t
.IR mutRate ]
.RB [ \-p
.IR varThres ]
.RB [ \-P
.IR prior ]
.RB [ \-1
.IR nGroup1 ]
.RB [ \-d
.IR minFrac ]
.RB [ \-U
.IR nPerm ]
.RB [ \-X
.IR permThres ]
.RB [ \-T
.IR trioType ]
.I in.bcf
.RI [ region ]
Convert between BCF and VCF, call variant candidates and estimate allele
frequencies.
.RS
.TP
.B Input/Output Options:
.TP 10
.B -A
Retain all possible alternate alleles at variant sites. By default, the view
command discards unlikely alleles.
.TP 10
.B -b
Output in the BCF format. The default is VCF.
.TP
.BI -D \ FILE
Sequence dictionary (list of chromosome names) for VCF->BCF conversion [null]
.TP
.B -F
Indicate PL is generated by r921 or before (ordering is different).
.TP
.B -G
Suppress all individual genotype information.
.TP
.BI -l \ FILE
List of sites at which information are outputted [all sites]
.TP
.B -N
Skip sites where the REF field is not A/C/G/T
.TP
.B -Q
Output the QCALL likelihood format
.TP
.BI -s \ FILE
List of samples to use. The first column in the input gives the sample names
and the second gives the ploidy, which can only be 1 or 2. When the 2nd column
is absent, the sample ploidy is assumed to be 2. In the output, the ordering of
samples will be identical to the one in
.IR FILE .
[null]
.TP
.B -S
The input is VCF instead of BCF.
.TP
.B -u
Uncompressed BCF output (force -b).
.TP
.B Consensus/Variant Calling Options:
.TP 10
.B -c
Call variants using Bayesian inference. This option automatically invokes option
.BR -e .
.TP
.BI -d \ FLOAT
When
.B -v
is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0]
.TP
.B -e
Perform max-likelihood inference only, including estimating the site allele frequency,
testing Hardy-Weinberg equlibrium and testing associations with LRT.
.TP
.B -g
Call per-sample genotypes at variant sites (force -c)
.TP
.BI -i \ FLOAT
Ratio of INDEL-to-SNP mutation rate [0.15]
.TP
.BI -p \ FLOAT
A site is considered to be a variant if P(ref|D)<FLOAT [0.5]
.TP
.BI -P \ STR
Prior or initial allele frequency spectrum. If STR can be
.IR full ,
.IR cond2 ,
.I flat
or the file consisting of error output from a previous variant calling
run.
.TP
.BI -t \ FLOAT
Scaled muttion rate for variant calling [0.001]
.TP
.BI -T \ STR
Enable pair/trio calling. For trio calling, option
.B -s
is usually needed to be applied to configure the trio members and their ordering.
In the file supplied to the option
.BR -s ,
the first sample must be the child, the second the father and the third the mother.
The valid values of
.I STR
are `pair', `trioauto', `trioxd' and `trioxs', where `pair' calls differences between two input samples, and `trioxd' (`trioxs') specifies that the input
is from the X chromosome non-PAR regions and the child is a female (male). [null]
.TP
.B -v
Output variant sites only (force -c)
.TP
.B Contrast Calling and Association Test Options:
.TP
.BI -1 \ INT
Number of group-1 samples. This option is used for dividing the samples into
two groups for contrast SNP calling or association test.
When this option is in use, the following VCF INFO will be outputted:
PC2, PCHI2 and QCHI2. [0]
.TP
.BI -U \ INT
Number of permutations for association test (effective only with
.BR -1 )
[0]
.TP
.BI -X \ FLOAT
Only perform permutations for P(chi^2)<FLOAT (effective only with
.BR -U )
[0.01]
.RE
.TP
.B index
.B bcftools index
.I in.bcf
Index sorted BCF for random access.
.RE
.TP
.B cat
.B bcftools cat
.I in1.bcf
.RI [ "in2.bcf " [ ... "]]]"
Concatenate BCF files. The input files are required to be sorted and
have identical samples appearing in the same order.
.RE
.SH SAM FORMAT
Sequence Alignment/Map (SAM) format is TAB-delimited. Apart from the header lines, which are started
with the `@' symbol, each alignment line consists of:
.TS
center box;
cb | cb | cb
n | l | l .
Col Field Description
_
1 QNAME Query template/pair NAME
2 FLAG bitwise FLAG
3 RNAME Reference sequence NAME
4 POS 1-based leftmost POSition/coordinate of clipped sequence
5 MAPQ MAPping Quality (Phred-scaled)
6 CIAGR extended CIGAR string
7 MRNM Mate Reference sequence NaMe (`=' if same as RNAME)
8 MPOS 1-based Mate POSistion
9 TLEN inferred Template LENgth (insert size)
10 SEQ query SEQuence on the same strand as the reference
11 QUAL query QUALity (ASCII-33 gives the Phred base quality)
12+ OPT variable OPTional fields in the format TAG:VTYPE:VALUE
.TE
.PP
Each bit in the FLAG field is defined as:
.TS
center box;
cb | cb | cb
l | c | l .
Flag Chr Description
_
0x0001 p the read is paired in sequencing
0x0002 P the read is mapped in a proper pair
0x0004 u the query sequence itself is unmapped
0x0008 U the mate is unmapped
0x0010 r strand of the query (1 for reverse)
0x0020 R strand of the mate
0x0040 1 the read is the first read in a pair
0x0080 2 the read is the second read in a pair
0x0100 s the alignment is not primary
0x0200 f the read fails platform/vendor quality checks
0x0400 d the read is either a PCR or an optical duplicate
.TE
where the second column gives the string representation of the FLAG field.
.SH VCF FORMAT
The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:
.TS
center box;
cb | cb | cb
n | l | l .
Col Field Description
_
1 CHROM CHROMosome name
2 POS the left-most POSition of the variant
3 ID unique variant IDentifier
4 REF the REFerence allele
5 ALT the ALTernate allele(s), separated by comma
6 QUAL variant/reference QUALity
7 FILTER FILTers applied
8 INFO INFOrmation related to the variant, separated by semi-colon
9 FORMAT FORMAT of the genotype fields, separated by colon (optional)
10+ SAMPLE SAMPLE genotypes and per-sample information (optional)
.TE
.PP
The following table gives the
.B INFO
tags used by samtools and bcftools.
.TS
center box;
cb | cb | cb
l | l | l .
Tag Format Description
_
AF1 double Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele
DP int Raw read depth (without quality filtering)
DP4 int[4] # high-quality reference forward bases, ref reverse, alternate for and alt rev bases
FQ int Consensus quality. Positive: sample genotypes different; negative: otherwise
MQ int Root-Mean-Square mapping quality of covering reads
PC2 int[2] Phred probability of AF in group1 samples being larger (,smaller) than in group2
PCHI2 double Posterior weighted chi^2 P-value between group1 and group2 samples
PV4 double[4] P-value for strand bias, baseQ bias, mapQ bias and tail distance bias
QCHI2 int Phred-scaled PCHI2
RP int # permutations yielding a smaller PCHI2
CLR int Phred log ratio of genotype likelihoods with and without the trio/pair constraint
UGT string Most probable genotype configuration without the trio constraint
CGT string Most probable configuration with the trio constraint
.TE
.SH EXAMPLES
.IP o 2
Import SAM to BAM when
.B @SQ
lines are present in the header:
samtools view -bS aln.sam > aln.bam
If
.B @SQ
lines are absent:
samtools faidx ref.fa
samtools view -bt ref.fa.fai aln.sam > aln.bam
where
.I ref.fa.
D0CA
fai
is generated automatically by the
.B faidx
command.
.IP o 2
Attach the
.B RG
tag while merging sorted alignments:
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
samtools merge -rh rg.txt merged.bam ga.bam 454.bam
The value in a
.B RG
tag is determined by the file name the read is coming from. In this
example, in the
.IR merged.bam ,
reads from
.I ga.bam
will be attached
.IR RG:Z:ga ,
while reads from
.I 454.bam
will be attached
.IR RG:Z:454 .
.IP o 2
Call SNPs and short INDELs for one diploid individual:
samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 > var.flt.vcf
The
.B -D
option of varFilter controls the maximum read depth, which should be
adjusted to about twice the average read depth. One may consider to add
.B -C50
to
.B mpileup
if mapping quality is overestimated for reads containing excessive
mismatches. Applying this option usually helps
.B BWA-short
but may not other mappers.
.IP o 2
Generate the consensus sequence for one diploid individual:
samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq
.IP o 2
Call somatic mutations from a pair of samples:
samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf
In the output INFO field,
.I CLR
gives the Phred-log ratio between the likelihood by treating the
two samples independently, and the likelihood by requiring the genotype to be identical.
This
.I CLR
is effectively a score measuring the confidence of somatic calls. The higher the better.
.IP o 2
Call de novo and somatic mutations from a family trio:
samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf
File
.I samples.txt
should consist of three lines specifying the member and order of samples (in the order of child-father-mother).
Similarly,
.I CLR
gives the Phred-log likelihood ratio with and without the trio constraint.
.I UGT
shows the most likely genotype configuration without the trio constraint, and
.I CGT
gives the most likely genotype configuration satisfying the trio constraint.
.IP o 2
Phase one individual:
samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out
The
.B calmd
command is used to reduce false heterozygotes around INDELs.
.IP o 2
Call SNPs and short indels for multiple diploid individuals:
samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf
bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf
Individuals are identified from the
.B SM
tags in the
.B @RG
header lines. Individuals can be pooled in one alignment file; one
individual can also be separated into multiple files. The
.B -P
option specifies that indel candidates should be collected only from
read groups with the
.B @RG-PL
tag set to
.IR ILLUMINA .
Collecting indel candidates from reads sequenced by an indel-prone
technology may affect the performance of indel calling.
.IP o 2
Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals:
samtools mpileup -Igf ref.fa *.bam > all.bcf
bcftools view -bl sites.list all.bcf > sites.bcf
bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs
bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs
bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs
......
where
.I sites.list
contains the list of sites with each line consisting of the reference
sequence name and position. The following
.B bcftools
commands estimate AFS by EM.
.IP o 2
Dump BAQ applied alignment for other SNP callers:
samtools calmd -bAr aln.bam > aln.baq.bam
It adds and corrects the
.B NM
and
.B MD
tags at the same time. The
.B calmd
command also comes with the
.B -C
option, the same as the one in
.B pileup
and
.BR mpileup .
Apply if it helps.
.SH LIMITATIONS
.PP
.IP o 2
Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
.IP o 2
Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan
reads or ends mapped to different chromosomes). If this is a concern,
please use Picard's MarkDuplicate which correctly handles these cases,
although a little slower.
.SH AUTHOR
.PP
Heng Li from the Sanger Institute wrote the C version of samtools. Bob
Handsaker from the Broad Institute implemented the BGZF library and Jue
Ruan from Beijing Genomics Institute wrote the RAZF library. John
Marshall and Petr Danecek contribute to the source code and various
people from the 1000 Genomes Project have contributed to the SAM format
specification.
.SH SEE ALSO
.PP
Samtools website: <http://samtools.sourceforge.net>