SAMTOOLS使用 SAM BAM文件处理

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samtools

高通量测序

比对

杂谈

分类： 生物信息

01

Usage: samtools view [options] <in.bam>|<in.sam> [region1 [...]]

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默认情况下不加 region，则是输出所有的 region.

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Options: -b       output BAM

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                  默认下输出是 SAM 格式文件，该参数设置输出 BAM 格式

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         -h       print header for the SAM output

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                  默认下输出的 sam 格式文件不带 header，该参数设定输出sam文件时带 header 信息

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         -H       print header only (no alignments)

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         -S       input is SAM

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                  默认下输入是 BAM 文件，若是输入是 SAM 文件，则最好加该参数，否则有时候会报错。

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         -u       uncompressed BAM output (force -b)

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                  该参数的使用需要有-b参数，能节约时间，但是需要更多磁盘空间。

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         -c       Instead of printing the alignments, only count them and print the

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                  total number. All filter options, such as ‘-f’, ‘-F’ and ‘-q’ ,

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                  are taken into account.

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         -1       fast compression (force -b)

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         -x       output FLAG in HEX (samtools-C specific)

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         -X       output FLAG in string (samtools-C specific)

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         -c       print only the count of matching records

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         -L FILE  output alignments overlapping the input BED FILE [null]

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         -t FILE  list of reference names and lengths (force -S) [null]

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                  使用一个list文件来作为header的输入

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         -T FILE  reference sequence file (force -S) [null]

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                  使用序列fasta文件作为header的输入

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         -o FILE  output file name [stdout]

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         -R FILE  list of read groups to be outputted [null]

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         -f INT   required flag, 0 for unset [0]

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         -F INT   filtering flag, 0 for unset [0]

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                  Skip alignments with bits present in INT [0]

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                  数字4代表该序列没有比对到参考序列上

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                  数字8代表该序列的mate序列没有比对到参考序列上

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         -q INT   minimum mapping quality [0]

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         -l STR   only output reads in library STR [null]

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         -r STR   only output reads in read group STR [null]

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         -s FLOAT fraction of templates to subsample; integer part as seed [-1]

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         -?       longer help

01

#提取比对到参考序列上的比对结果

02

$ samtools view -bF 4 abc.bam > abc.F.bam

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#提取paired reads中两条reads都比对到参考序列上的比对结果，只需要把两个4+8的值12作为过滤参数即可

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$ samtools view -bF 12 abc.bam > abc.F12.bam

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#提取没有比对到参考序列上的比对结果

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$ samtools view -bf 4 abc.bam > abc.f.bam

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#提取bam文件中比对到caffold1上的比对结果，并保存到sam文件格式

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$ samtools view abc.bam scaffold1 > scaffold1.sam

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#提取scaffold1上能比对到30k到100k区域的比对结果

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$ samtools view abc.bam scaffold1:30000-100000 > scaffold1_30k-100k.sam

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#根据fasta文件，将 header 加入到 sam 或 bam 文件中

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$ samtools view -T genome.fasta -h scaffold1.sam > scaffold1.h.sam

1

Usage: samtools sort [-n] [-m <maxMem>] <in.bam> <out.prefix> 

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-m 参数默认下是 500,000,000 即500M（不支持K，M，G等缩写）。对于处理大数据时，如果内存够用，则设置大点的值，以节约时间。

3

-n 设定排序方式按short reads的ID排序。默认下是按序列在fasta文件中的顺序（即header）和序列从左往右的位点排序。

01

Usage:   samtools merge [-nr] [-h inh.sam] <out.bam> <in1.bam> <in2.bam>[...]

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Options: -n       sort by read names

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         -r       attach RG tag (inferred from file names)

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         -u       uncompressed BAM output

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         -f       overwrite the output BAM if exist

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         -1       compress level 1

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         -R STR   merge file in the specified region STR [all]

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         -h FILE  copy the header in FILE to <out.bam> [in1.bam]

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Note: Samtools' merge does not reconstruct the @RG dictionary in the header. Users

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      must provide the correct header with -h, or uses Picard which properly maintains

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      the header dictionary in merging.

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#以下两种命令结果一样

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<span style="font-weight: inherit; font-style: inherit; color: #ff6600;">$ samtools index abc.sort.bam</span>

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$ samtools index abc.sort.bam abc.sort.bam.bai

1

Usage: samtools faidx <in.bam> [ [...]]

2

3

对基因组文件建立索引

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$ samtools faidx genome.fasta

5

#生成了索引文件genome.fasta.fai,是一个文本文件，分成了5列。第一列是子序列的名称；第二列是子序列的长度；个人认为“第三列是序列所在的位置”，因为该数字从上往下逐渐变大，最后的数字是genome.fasta文件的大小；第4和5列不知是啥意思。于是通过此文件，可以定位子序列在fasta文件在磁盘上的存放位置，直接快速调出子序列。

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#由于有索引文件，可以使用以下命令很快从基因组中提取到fasta格式的子序列

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$ samtools faidx genome.fasta scffold_10 > scaffold_10.fasta

01

Usage: samtools tview <aln.bam> [ref.fasta]

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当给出参考基因组的时候，会在第一排显示参考基因组的序列，否则，第一排全用N表示。

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按下 g ，则提示输入要到达基因组的某一个位点。例子“scaffold_10:1000"表示到达第

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10号scaffold的第1000个碱基位点处。

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使用H(左）J（上）K（下）L（右）移动显示界面。大写字母移动快，小写字母移动慢。

07

使用空格建向左快速移动（和 L 类似），使用Backspace键向左快速移动（和 H 类似）。

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Ctrl+H 向左移动1kb碱基距离； Ctrl+L 向右移动1kb碱基距离

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可以用颜色标注比对质量，碱基质量，核苷酸等。30～40的碱基质量或比对质量使用白色表示；

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20～30黄色；10～20绿色；0～10蓝色。

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使用点号'.'切换显示碱基和点号；使用r切换显示read name等

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还有很多其它的使用说明，具体按 ？ 键来查看。

01

Usage: samtools flagstat <in.bam>

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$ samtools flagstat example.bam

04

11945742 + 0 in total (QC-passed reads + QC-failed reads)

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#总共的reads数

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0 + 0 duplicates

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7536364 + 0 mapped (63.09%:-nan%)

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#总体上reads的匹配率

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11945742 + 0 paired in sequencing

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#有多少reads是属于paired reads

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5972871 + 0 read1

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#reads1中的reads数

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5972871 + 0 read2

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#reads2中的reads数

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6412042 + 0 properly paired (53.68%:-nan%)

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#完美匹配的reads数：比对到同一条参考序列，并且两条reads之间的距离符合设置的阈值

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6899708 + 0 with itself and mate mapped

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#paired reads中两条都比对到参考序列上的reads数

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636656 + 0 singletons (5.33%:-nan%)

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#单独一条匹配到参考序列上的reads数，和上一个相加，则是总的匹配上的reads数。

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469868 + 0 with mate mapped to a different chr

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#paired reads中两条分别比对到两条不同的参考序列的reads数

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243047 + 0 with mate mapped to a different chr (mapQ>=5)

1

Usage: bam2depth [-r reg] [-q baseQthres] [-Q mapQthres] [-b in.bed] <in1.bam> [...]

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$ samtools reheader <in.header.sam> <in.bam>

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$ samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ]

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$ samtools idxstats <aln.bam>

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$ wget http://www.hudsonalpha.org/gsl/static/software/bam2fastq-1.1.0.tgz

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$ tar zxf bam2fastq-1.1.0.tgz

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$ cd bam2fastq-1.1.0

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$ make

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$ ./bam2fastq <in.bam>

1

Usage: samtools mpileup [-EBug] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-M capMapQ] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]

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$ samtools mpileup -f genome.fasta abc.bam > abc.txt

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$ samtools mpileup -gSDf genome.fasta abc.bam > abc.bcf

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$ samtools mpileup -guSDf genome.fasta abc.bam |

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 bcftools view -cvNg - > abc.vcf

01

scaffold_1      2841    A       11      ,,,...,....     BHIGDGIJ?FF

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scaffold_1      2842    C       12      ,$,,...,....^I. CFGEGEGGCFF+

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scaffold_1      2843    G       11      ,,...,.....     FDDDDCD?DD+

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scaffold_1      2844    G       11      ,,...,.....     FA?AAAA<AA+

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scaffold_1      2845    G       11      ,,...,.....     F656666166*

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scaffold_1      2846    A       11      ,,...,.....     (1.1111)11*

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scaffold_1      2847    A       11      ,,+9acggtgaag.+9ACGGTGAAT.+9ACGGTGAAG.+9ACGGTGAAG,+9acggtgaag.+9ACGGTGAAG.+9ACGGTGAAG.+9ACGGTGAAG.+9ACGGTGAAG.+9ACGGTGAAG       %.+....-..)

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scaffold_1      2848    N       11      agGGGgGGGGG     !!$!!!!!!!!

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scaffold_1      2849    A       11      c$,...,.....    !0000000000

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scaffold_1      2850    A       10      ,...,.....      353333333

01

#输入参数

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-6 Assume the quality is in the Illumina 1.3+ encoding. -A Do not skip anomalous read pairsin variant calling.

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-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.

04

-b FILE List of input BAM files, one file per line [null]

05

-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]

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-d INT At a position, read maximally INT reads per input BAM. [250]

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-E Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt specificity a little bit.

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-f FILE The faidx-indexed reference file in the FASTA format. The file can be optionally compressed by razip. [null]

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-l FILE BED or position list file containing a list of regions or sites where pileup or BCF should be generated [null]

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-M INT       cap mapping quality at INT [60]

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-q INT Minimum mapping quality for an alignment to be used [0]

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-Q INT Minimum base quality for a base to be considered [13]

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-r STR Only generate pileup in region STR [all sites]

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输出参数

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-D Output per-sample read depth (require -g/-u)

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-g Compute genotype likelihoods and output them in the binary call format (BCF).

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-S Output per-sample Phred-scaled strand bias P-value (require -g/-u)

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-u Similar to -g except that the output is uncompressed BCF, which is preferred for piping.

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Options for Genotype Likelihood Computation (for -g or -u):

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-e INT Phred-scaled gap extension sequencing error probability. Reducing INT leads to longer indels. [20]

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-h INT Coefficient for modeling homopolymer errors. Given an l-long homopolymer run, the sequencing error of an indel of size s is modeled as INT*s/l. [100]

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-I Do not perform INDEL calling

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-L INT Skip INDEL calling if the average per-sample depth is above INT. [250]

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-o INT Phred-scaled gap open sequencing error probability. Reducing INT leads to more indel calls. [40]

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-P STR Comma dilimited list of platforms (determined by @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]

1

$ bcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s listSample]

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          [-i gapSNPratio] [-t mutRate] [-p varThres] [-P prior]

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          [-1 nGroup1] [-d minFrac] [-U nPerm] [-X permThres]

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          [-T trioType] in.bcf [region]

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$ bcftools view -cvNg abc.bcf > snp_indel.vcf

1

scaffold_1      2847    .       A       AACGGTGAAG      194     .       INDEL;DP=11;VDB=0.0401;AF1=1;AC1=2;DP4=0,0,8,3;MQ=35;FQ=-67.5   GT:PL:GQ        1/1:235,33,0:63

2

scaffold_1      3908    .       G       A       111     .       DP=13;VDB=0.0085;AF1=1;AC1=2;DP4=0,0,5,7;MQ=42;FQ=-63   GT:PL:GQ        1/1:144,36,0:69

3

scaffold_1      4500    .       A       G       31.5    .       DP=8;VDB=0.0034;AF1=1;AC1=2;DP4=0,0,1,3;MQ=42;FQ=-39    GT:PL:GQ        1/1:64,12,0:21

4

scaffold_1      4581    .       TGGNGG  TGG     145     .       INDEL;DP=8;VDB=0.0308;AF1=1;AC1=2;DP4=0,0,0,8;MQ=42;FQ=-58.5    GT:PL:GQ        1/1:186,24,0:45

5

scaffold_1      4644    .       G       A       195     .       DP=21;VDB=0.0198;AF1=1;AC1=2;DP4=0,0,10,10;MQ=42;FQ=-87 GT:PL:GQ        1/1:228,60,0:99

6

scaffold_1      4827    .       NACAAAGA        NA      4.42    .       INDEL;DP=1;AF1=1;AC1=2;DP4=0,0,1,0;MQ=40;FQ=-37.5       GT:PL:GQ        0/1:40,3,0:3

7

scaffold_1      4854    .       A       G       48      .       DP=6;VDB=0.0085;AF1=1;AC1=2;DP4=0,0,2,1;MQ=41;FQ=-36    GT:PL:GQ        1/1:80,9,0:16

8

scaffold_1      5120    .       A       G       85      .       DP=8;VDB=0.0355;AF1=1;AC1=2;DP4=0,0,5,3;MQ=42;FQ=-51    GT:PL:GQ        1/1:118,24,0:45

01

Tag   Format   Description

02

AF1   double   Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele

03

DP int   Raw read depth (without quality filtering)

04

DP4   int[4]   # high-quality reference forward bases, ref reverse, alternate for and alt rev bases

05

FQ int   Consensus quality. Positive: sample genotypes different; negative: otherwise

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MQ int   Root-Mean-Square mapping quality of covering reads

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PC2   int[2]   Phred probability of AF in group1 samples being larger (,smaller) than ingroup2

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PCHI2 double   Posterior weighted chi^2 P-value between group1 and group2 samples

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PV4   double[4]   P-value for strand bias, baseQ bias, mapQ bias and tail distance bias

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QCHI2 int   Phred-scaled PCHI2

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RP int   # permutations yielding a smaller PCHI2

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CLR   int   Phred log ratio of genotype likelihoods with and without the trio/pair constraint

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UGT   string   Most probable genotype configuration without the trio constraint

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CGT   string   Most probable configuration with the trio constraint

01

Input/Output Options:

02

-A Retain all possible alternate alleles at variant sites. By default, the view commanddiscards unlikely alleles.

03

-b Output in the BCF format. The default is VCF.

04

-D FILE Sequence dictionary (list of chromosome names) for VCF->BCF conversion [null]

05

-F Indicate PL is generated by r921 or before (ordering is different).

06

-G Suppress all individual genotype information.

07

-l FILE List of sites at which information are outputted [all sites]

08

-N Skip sites where the REF field is not A/C/G/T

09

-Q Output the QCALL likelihood format

10

-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 FILE. [null]

11

-S The input is VCF instead of BCF.

12

-u Uncompressed BCF output (force -b).

13

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Consensus/Variant Calling Options:

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-c Call variants using Bayesian inference. This option automatically invokes option -e.

16

-d FLOAT When -v is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0]

17

        当有多个sample用于variants calling时，比如多个转录组数据或多个重测序

18

        数据需要比对到参考基因组上，设置该值，表明至少有该<float 0~1>比例的

19

        samples在该位点都有覆盖才计算入variant.所以对于只有一个sample的情况

20

        下，该值设置在0～1之间没有意义，大于1则得不到任何结果。

21

-e Perform max-likelihood inference only, including estimating the site allele frequency, testing Hardy-Weinberg equlibrium and testing associations with LRT.

22

-g Call per-sample genotypes at variant sites (force -c)

23

-i FLOAT Ratio of INDEL-to-SNP mutation rate [0.15]

24

-p FLOAT A site is considered to be a variant if P(ref|D)

25

-t FLOAT Scaled muttion rate for variant calling [0.001]

26

-T STR Enable pair/trio calling. For trio calling, option -s is usually needed to be applied to configure the trio members and their ordering. In the file supplied to the option -s, the first sample must be the child, the second the father and the third the mother. The valid values of 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]

27

-v Output variant sites only (force -c)

28

29

Contrast Calling and Association Test Options:

30

-1 INT   Number of group-1 samples. This option is used for dividing the samples into twogroups 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]

31

-U INT   Number of permutations for association test (effective only with -1) [0]

32

-X FLOAT Only perform permutations for P(chi^2)

1

$ perl -ne 'print $_ if /DP4=(d+),(d+),(d+),(d+)/ && ($3+$4)>=10 && ($3+$4)/($1+$2+$3+$4)>=0.8' snp_indel.vcf > snp_indel.final.vcf

1

Usage:  samtools rmdup [-sS] 

2

-s 对single-end reads。默认情况下，只对paired-end reads

3

-S 将Paired-end reads作为single-end reads处理。

4

5

$ samtools input.sorted.bam output.bam

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.

OPTIONS:

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.

这个命令是查看的命令，看到的是map以后覆盖度的文件，samtools tview .bam文件 .ref文件

mpileup

samtools mpileup [-EBug] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-M capMapQ][-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]

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 -uor-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.

Input Options:

reheader

samtools reheader <in.header.sam> <in.bam>

Replace the header in in.bam with the header in in.header.sam. This command is much faster than replacing the header with a BAM->SAM->BAM conversion.

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 toreheader which enables fast BAM concatenation.

sort

samtools sort [-no] [-m maxMem] <in.bam> <out.prefix>

Sort alignments by leftmost coordinates. File <out.prefix>.bam will be created. This command may also create temporary files <out.prefix>.%d.bam when the whole alignment cannot be fitted into memory (controlled by option -m).

OPTIONS:

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 inh.sam, if any, must all refer to the same set of reference sequences. The header reference list and (unless overridden by -h) ‘@’ headers of in1.bam will be copied to out.bam, and the headers of other files will be ignored.

OPTIONS:

index

samtools index <aln.bam>

Index sorted alignment for fast random access. Index file <aln.bam>.bai will be created.

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.

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, faidx will index the file and create<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 RAZF format.

fixmate

samtools fixmate <in.nameSrt.bam> <out.bam>

Fill in mate coordinates, ISIZE and mate related flags from a name-sorted alignment.

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 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).

OPTIONS:

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.

OPTIONS:

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 -f is in use, BAQ will be applied. This command is only designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].

-b

Output in the BAM format.我们第一步把sam转换成bam的中-bS中-b表示的就是要输出bam的文件

-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]

-F INT

Skip alignments with bits present in INT [0]

-h

Include the header in the output.（再输出文件中包含头文件）

-H

Output the header only.（只输出头文件）

-l STR

Only output reads in library STR [null]

-o FILE

Output file [stdout]

-q INT

Skip alignments with MAPQ smaller than INT [0]

-r STR

Only output reads in read group STR [null]

-R FILE

Output reads in read groups listed in FILE [null]

-S

Input is in SAM. If @SQ header lines are absent, the ‘-t’ option is required.这里S表示的就是输入的是SAM的格式，如果sam中没有头文件，那么就要用到-t的选项

-c

Instead of printing the alignments, only count them and print the total number. All filter options, such as ‘-f’, ‘-F’ and ‘-q’ , are taken into account.

-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 <ref.fa>.fai can be used as this <in.ref_list> file.

-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.

-6

Assume the quality is in the Illumina 1.3+ encoding. -A Do not skip anomalous read pairs in variant calling.

-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.

-b FILE

List of input BAM files, one file per line [null]

-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]

-d INT

At a position, read maximally INT reads per input BAM. [250]

-E

Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt specificity a little bit.

-f FILE

The faidx-indexed reference file in the FASTA format. The file can be optionally compressed by razip. [null]：要有一个参考序列

-l FILE

BED or position list file containing a list of regions or sites where pileup or BCF should be generated [null]

-q INT

Minimum mapping quality for an alignment to be used [0]

-Q INT

Minimum base quality for a base to be considered [13]

-r STR

Only generate pileup in region STR [all sites]

Output Options:输出选项

-D

Output per-sample read depth 读取的深度，可以设定值比如-D100

-g

Compute genotype likelihoods and output them in the binary call format (BCF).

-S

Output per-sample Phred-scaled strand bias P-value

-u

Similar to -g except that the output is uncompressed（未压缩的） BCF, which is preferred for piping.

Options for Genotype Likelihood Computation (for -g or -u):

-e INT

Phred-scaled gap extension sequencing error probability. Reducing INTleads to longer indels. [20]

-h INT

Coefficient for modeling homopolymer errors. Given an l-long homopolymer run, the sequencing error of an indel of size s is modeled as INT*s/l. [100]

-I

Do not perform INDEL calling

-L INT

Skip INDEL calling if the average per-sample depth is above INT. [250]

-o INT

Phred-scaled gap open sequencing error probability. Reducing INT leads to more indel calls. [40]

-P STR

Comma dilimited list of platforms (determined by @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]

-o

Output the final alignment to the standard output.

-n

Sort by read names rather than by chromosomal coordinates

-m INT

Approximately the maximum required memory. [500000000]

-1

Use zlib compression level 1 to comrpess the output

-f

Force to overwrite the output file if present.

-h FILE

Use the lines of FILE as ‘@’ headers to be copied to out.bam, replacing any header lines that would otherwise be copied from in1.bam. (FILE is actually in SAM format, though any alignment records it may contain are ignored.)

-n

The input alignments are sorted by read names rather than by chromosomal coordinates

-R STR

Merge files in the specified region indicated by STR [null]

-r

Attach an RG tag to each alignment. The tag value is inferred from file names.

-u

Uncompressed BAM output

-s

Remove duplicate for single-end reads. By default, the command works for paired-end reads only.

-S

Treat paired-end reads and single-end reads.

-A

When used jointly with -r this option overwrites the original base quality.

-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.

-u

Output uncompressed BAM

-b

Output compressed BAM

-S

The input is SAM with header lines

-C INT

Coefficient to cap mapping quality of poorly mapped reads. See the pileupcommand for details. [0]

-r

Compute the BQ tag (without -A) or cap base quality by BAQ (with -A).

-E

Extended BAQ calculation. This option trades specificity for sensitivity, though the effect is minor.

phase

samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam>

Call and phase heterozygous SNPs. OPTIONS:

-A

Drop reads with ambiguous phase.

-b STR

Prefix of BAM output. When this option is in use, phase-0 reads will be saved in fileSTR.0.bam and phase-1 reads in STR.1.bam. Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads with switch errors will be saved inSTR.chimeric.bam. [null]

-F

Do not attempt to fix chimeric reads.

-k INT

Maximum length for local phasing. [13]

-q INT

Minimum Phred-scaled LOD to call a heterozygote. [40]

-Q INT

Minimum base quality to be used in het calling. [13]

view

bcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s listSample] [-igapSNPratio] [-t mutRate] [-p varThres] [-P prior] [-1 nGroup1] [-d minFrac] [-UnPerm] [-X permThres] [-T trioType] in.bcf [region]

Convert between BCF and VCF, call variant candidates and estimate allele frequencies.

index

bcftools index in.bcf

Index sorted BCF for random access.

Input/Output Options: 

-A

Retain all possible alternate alleles at variant sites. By default, the view command discards unlikely alleles.

-b

Output in the BCF format. The default is VCF.

-D FILE

Sequence dictionary (list of chromosome names) for VCF->BCF conversion [null]

-F

Indicate PL is generated by r921 or before (ordering is different).

-G

Suppress all individual genotype information.

-l FILE

List of sites at which information are outputted [all sites]

-N

Skip sites where the REF field is not A/C/G/T

-Q

Output the QCALL likelihood format

-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 FILE. [null]

-S

The input is VCF instead of BCF.

-u

Uncompressed BCF output (force -b).

Consensus/Variant Calling Options: 

-c

Call variants using Bayesian inference. This option automatically invokes option -e.

-d FLOAT

When -v is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0]

-e

Perform max-likelihood inference only, including estimating the site allele frequency, testing Hardy-Weinberg equlibrium and testing associations with LRT.

-g

Call per-sample genotypes at variant sites (force -c)

-i FLOAT

Ratio of INDEL-to-SNP mutation rate [0.15]

-p FLOAT

A site is considered to be a variant if P(ref|D)<FLOAT [0.5]

-P STR

Prior or initial allele frequency spectrum. If STR can be full, cond2,flat or the file consisting of error output from a previous variant calling run.

-t FLOAT

Scaled muttion rate for variant calling [0.001]

-T STR

Enable pair/trio calling. For trio calling, option -s is usually needed to be applied to configure the trio members and their ordering. In the file supplied to the option -s, the first sample must be the child, the second the father and the third the mother. The valid values of 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]

-v

Output variant sites only (force -c)：这里有-v这个选项就只输出snp位点，如果没有-v那么就是输出所有的位点（测序所包含的）

Contrast Calling and Association Test Options: 

-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]

-U INT

Number of permutations for association test (effective only with -1) [0]

-X FLOAT

Only perform permutations for P(chi^2)<FLOAT (effective only with -U) [0.01]

cat

bcftools cat in1.bcf ["in2.bcf "[..."]]]"

Concatenate BCF files. The input files are required to be sorted and have identical samples appearing in the same order.

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

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

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)

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

o

Import SAM to BAM when @SQ lines are present in the header:

samtools view -bS aln.sam > aln.bam

If @SQ lines are absent:

samtools faidx ref.fa 

samtools view -bt ref.fa.fai aln.sam > aln.bam

where ref.fa.fai is generated automatically by the faidx command.

o

Attach the RG tag while merging sorted alignments:

perl -e ’print "@RG ID:ga SM:hs LB:ga PL:Illumina @RG ID:454 SM:hs LB:454 PL:454 "’ > rg.txt 

samtools merge -rh rg.txt merged.bam ga.bam 454.bam

The value in a RG tag is determined by the file name the read is coming from. In this example, in the merged.bam, reads from ga.bam will be attached RG:Z:ga, while reads from454.bam will be attached RG:Z:454.

o

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 -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 -C50 to mpileup if mapping quality is overestimated for reads containing excessive mismatches. Applying this option usually helps BWA-short but may not other mappers.

o

Generate the consensus sequence for one diploid individual:

samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq

o

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, 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 CLR is effectively a score measuring the confidence of somatic calls. The higher the better.

o

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 samples.txt should consist of three lines specifying the member and order of samples (in the order of child-father-mother). Similarly, CLR gives the Phred-log likelihood ratio with and without the trio constraint. UGT shows the most likely genotype configuration without the trio constraint, and CGT gives the most likely genotype configuration satisfying the trio constraint.

o

Phase one individual:

samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out

The calmd command is used to reduce false heterozygotes around INDELs.

o

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 SM tags in the @RG header lines. Individuals can be pooled in one alignment file; one individual can also be separated into multiple files. The-P option specifies that indel candidates should be collected only from read groups with the @RG-PL tag set to ILLUMINA. Collecting indel candidates from reads sequenced by an indel-prone technology may affect the performance of indel calling.

o

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 sites.list contains the list of sites with each line consisting of the reference sequence name and position. The following bcftools commands estimate AFS by EM.

o

Dump BAQ applied alignment for other SNP callers:

samtools calmd -bAr aln.bam > aln.baq.bam

It adds and corrects the NM and MD tags at the same time. The calmd command also comes with the -C option, the same as the one in pileup and mpileup. Apply if it helps.

o

Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.

o

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.

samtools-0.1.17

samtools (1)

05 July 2011