SAMTOOLS使用 SAM BAM文件处理

【怪毛匠子 整理】

samtools学习及使用范例,以及官方文档详解

#第一步：把sam文件转换成bam文件,我们得到map.bam文件

system"samtools view -bS map.sam > map.bam";

#第二步：sort 一下 BAM 文件,得到map.sorted.bam

system"samtools sort map.b/am map.sorted";

#第三步：创建一个关于bam的索引文件,我们得到一个map.sorted.bam.bai的文件

system"samtools index map.sorted.bam";

#第四步：找snp，这里用的是sort以后的bam文件，如果不是，就会不断的报错

system"samtools mpileup -ugf TAIR10.fas map.sorted.bam | bcftools view -vcg -D100 ->snp.vcf"  //snp位点

perl 语言

如果我们要获取全部的位点的信息，而不是仅仅snp位点，那么我们只需要把最后一行的-v去掉就可以了

1. system"samtools mpileup -ugf TAIR10.fas map.sorted.bam | bcftools view -cg -D100 ->snp.vcf"  //所有位点

再下面有详细的解释-v的作用：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）

samtools 功能介绍

(2012-10-25 22:06:30)

标签： samtools 高通量测序 比对 杂谈

分类： 生物信息

samtools  输入bam文件，导出sam文件。同时可以进行排序，合并，建立索引等功能，并支持从特定区域内查找reads。

samtools 可以应用于linux的命令管道里。以“-”作为标准输入或输出。

view  从bam/sam文件中提取/打印部分比对结果。默认为所有的区域，也可以染色体区域（1-based，须sort并index）。

例如：

samtools view -bt ref_list.txt -o aln.bam aln.sam.gz

samtools view aln.sorted.bam chr2:20,100,000-20,200,000

tview

mpileup 列举每条reads比对的indel，SNP等信息。

“.”表示match;

“，”表示反向match;

“<”或“>”表示reference skip;

"ATCGN"表示正向mismatch;

"atcgn"表示反向mismatch;

‘\+[0-9]+[ACGTNacgtn]+’ insertion;

‘-[0-9]+[ACGTNacgtn]+’ 表示deletion;

“^”标记reads起始;

“$”标记reads segment结尾;

samtools pileup -vcf ref.fasta aln.sorted.bam

samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam

merge 合并bam文件

samtools merge out.bam in1.bam in2.bam in3.bam

tview 图形化的界面查看mapping的结果

? -help

g -到达指定的染色体区域

samtools tview aln.sorted.bam ref.fasta

sort 将比对结果排序

samtools sort aln.bam aln.sorted

reheader 将bam文件的头文件替换。

index 为sort后的bam文件建立所以，这是在 view 比对结果时指定染色体区域必须做的。

samtools index aln.sorted.bam

rmdup 去除可能的PCR重复。将比对到相同位置的reads去重，仅保留比对质量最好的一个。一般只对paired end reads（fr）有效，并须指定ISIZE。

phase Call and phase heterozygous SNPs

samtools常用命令详解

2014年01月26日 ⁄ Bioinformatics ⁄ 字号 小 中 大 ⁄ 评论 1 条 ⁄ 阅读 12,457 次 [点击加入在线收藏夹]

samtools的说明文档：http://samtools.sourceforge.net/samtools.shtml

samtools是一个用于操作sam和bam文件的工具合集。包含有许多命令。以下是常用命令的介绍

1. view

view命令的主要功能是：将sam文件转换成bam文件；然后对bam文件进行各种操作，比如数据的排序(不属于本命令的功能)和提取(这些操作是对bam文件进行的，因而当输入为sam文件的时候，不能进行该操作)；最后将排序或提取得到的数据输出为bam或sam（默认的）格式。

bam文件优点：bam文件为二进制文件，占用的磁盘空间比sam文本文件小；利用bam二进制文件的运算速度快。

view命令中，对sam文件头部的输入(-t或-T）和输出(-h)是单独的一些参数来控制的。

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

02	默认情况下不加 region，则是输出所有的 region.

03

04	Options: -b       output BAM

05	默认下输出是 SAM 格式文件，该参数设置输出 BAM 格式

06	-h       print header for the SAM output

07	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

08	-H       print header only (no alignments)

09	-S       input is SAM

10	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。

11	-u       uncompressed BAM output (force -b)

12	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。

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

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

15	are taken into account.

16	-1       fast compression (force -b)

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

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

19	-c       print only the count of matching records

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

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

22	使用一个list文件来作为header的输入

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

24	使用序列fasta文件作为header的输入

25	-o FILE  output file name [stdout]

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

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

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

29	Skip alignments with bits present in INT [0]

30	数字4代表该序列没有比对到参考序列上

31	数字8代表该序列的mate序列没有比对到参考序列上

32	-q INT   minimum mapping quality [0]

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

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

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

36	-?       longer help

例子：

#将sam文件转换成bam文件 $ samtools view -bS abc.sam > abc.bam $ samtools view -b -S abc.sam -o abc.bam

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

03

04	#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

05	$ samtools view -bF 12 abc.bam > abc.F12.bam

06

07	#提取没有比对到参考序列上的比对结果

08	$ samtools view -bf 4 abc.bam > abc.f.bam

09

10	#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

11	$ samtools view abc.bam scaffold1 > scaffold1.sam

12

13	#提取scaffold1上能比对到30k到100k区域的比对结果

14	$ samtools view abc.bam scaffold1:30000-100000 > scaffold1_30k-100k.sam

15

16	#根据fasta文件，将 header 加入到 sam 或 bam 文件中

17	$ samtools view -T genome.fasta -h scaffold1.sam > scaffold1.h.sam

2. sort

sort对bam文件进行排序。

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

2	-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。

例子：

$ samtools sort abc.bam abc.sort $ samtools view abc.sort.bam | less -S

3.merge

将2个或2个以上的已经sort了的bam文件融合成一个bam文件。融合后的文件不需要则是已经sort过了的。

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

02

03	Options: -n       sort by read names

04	-r       attach RG tag (inferred from file names)

05	-u       uncompressed BAM output

06	-f       overwrite the output BAM if exist

07	-1       compress level 1

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

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

10

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

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

13	the header dictionary in merging.

4.index

必须对bam文件进行默认情况下的排序后，才能进行index。否则会报错。

建立索引后将产生后缀为.bai的文件，用于快速的随机处理。很多情况下需要有bai文件的存在，特别是显示序列比对情况下。比如samtool的tview命令就需要；gbrowse2显示reads的比对图形的时候也需要。

Usage: samtools index <in.bam> [out.index]

例子：

1	#以下两种命令结果一样

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

3	$ samtools index abc.sort.bam abc.sort.bam.bai

5. faidx

对fasta文件建立索引,生成的索引文件以.fai后缀结尾。该命令也能依据索引文件快速提取fasta文件中的某一条（子）序列

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

2

3	对基因组文件建立索引

4	$ 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。

6

7	#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

8	$ samtools faidx genome.fasta scffold_10 > scaffold_10.fasta

6. tview

tview能直观的显示出reads比对基因组的情况，和基因组浏览器有点类似。

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

02

03	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。

04	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

05	10号scaffold的第1000个碱基位点处。

06	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）。

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

09	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；

10	20～30黄色；10～20绿色；0～10蓝色。

11	使用点号'.'切换显示碱基和点号；使用r切换显示read name等

12	还有很多其它的使用说明，具体按 ？ 键来查看。

7. flagstat

给出BAM文件的比对结果

01	Usage: samtools flagstat <in.bam>

02

03	$ samtools flagstat example.bam

04	11945742 + 0 in total (QC-passed reads + QC-failed reads)

05	#总共的reads数

06	0 + 0 duplicates

07	7536364 + 0 mapped (63.09%:-nan%)

08	#总体上reads的匹配率

09	11945742 + 0 paired in sequencing

10	#有多少reads是属于paired reads

11	5972871 + 0 read1

12	#reads1中的reads数

13	5972871 + 0 read2

14	#reads2中的reads数

15	6412042 + 0 properly paired (53.68%:-nan%)

16	#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

17	6899708 + 0 with itself and mate mapped

18	#paired reads中两条都比对到参考序列上的reads数

19	636656 + 0 singletons (5.33%:-nan%)

20	#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。

21	469868 + 0 with mate mapped to a different chr

22	#paired reads中两条分别比对到两条不同的参考序列的reads数

23	243047 + 0 with mate mapped to a different chr (mapQ>=5)

#同上一个，只是其中比对质量>=5的reads的数量

7. depth

得到每个碱基位点的测序深度,并输出到标准输出。

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

8. 其它有用的命令

reheader 替换bam文件的头

1	$ samtools reheader <in.header.sam> <in.bam>

cat 连接多个bam文件，适用于非sorted的bam文件

1	$ samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ]

idxstats 统计一个表格，4列，分别为”序列名，序列长度，比对上的reads数，unmapped reads number”。第4列应该是paired reads中有一端能匹配到该scaffold上，而另外一端不匹配到任何scaffolds上的reads数。

1	$ samtools idxstats <aln.bam>

9. 将bam文件转换为fastq文件

有时候，我们需要提取出比对到一段参考序列的reads，进行小范围的分析，以利于debug等。这时需要将bam或sam文件转换为fastq格式。

该网站提供了一个bam转换为fastq的程序：http://www.hudsonalpha.org/gsl/information/software/bam2fastq

1	$ wget http://www.hudsonalpha.org/gsl/static/software/bam2fastq-1.1.0.tgz

2	$ tar zxf bam2fastq-1.1.0.tgz

3	$ cd bam2fastq-1.1.0

4

$ make

5	$ ./bam2fastq <in.bam>

10. mpileup

samtools还有个非常重要的命令mpileup，以前为pileup。该命令用于生成bcf文件，再使用bcftools进行SNP和Indel的分析。bcftools是samtool中附带的软件，在samtools的安装文件夹中可以找到。

最常用的参数有2：

-f 来输入有索引文件的fasta参考序列；

-g 输出到bcf格式。用法和最简单的例子如下

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

2

3	$ samtools mpileup -f genome.fasta abc.bam > abc.txt

4	$ samtools mpileup -gSDf genome.fasta abc.bam > abc.bcf

5	$ samtools mpileup -guSDf genome.fasta abc.bam | \

6	bcftools view -cvNg - > abc.vcf

mpileup不使用-u或-g参数时，则不生成二进制的bcf文件，而生成一个文本文件(输出到标准输出)。该文本文件统计了参考序列中每个碱基位点的比对情况；该文件每一行代表了参考序列中某一个碱基位点的比对结果。比如：

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

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

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

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

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

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

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

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

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

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

mpileup生成的结果包含6行：参考序列名；位置；参考碱基；比对上的reads数；比对情况；比对上的碱基的质量。其中第5列比较复杂,解释如下：

1 ‘.’代表与参考序列正链匹配。

2 ‘,’代表与参考序列负链匹配。

3 ‘ATCGN’代表在正链上的不匹配。

4 ‘atcgn’代表在负链上的不匹配。

5 ‘*’代表模糊碱基

6 ‘^’代表匹配的碱基是一个read的开始；’^'后面紧跟的ascii码减去33代表比对质量；这两个符号修饰的是后面的碱基，其后紧跟的碱基(.,ATCGatcgNn)代表该read的第一个碱基。

7 ‘$’代表一个read的结束，该符号修饰的是其前面的碱基。

8 正则式’\+[0-9]+[ACGTNacgtn]+’代表在该位点后插入的碱基；比如上例中在scaffold_1的2847后插入了9个长度的碱基acggtgaag。表明此处极可能是indel。

9 正则式’-[0-9]+[ACGTNacgtn]+’代表在该位点后缺失的碱基；

pileup具体的参数如下：

01

#输入参数

02	-6 Assume the quality is in the Illumina 1.3+ encoding. -A Do not skip anomalous read pairsin variant calling.

03	-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]

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

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

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

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

10	-M INT       cap mapping quality at INT [60]

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

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

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

14

15

输出参数

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

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

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

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

20

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

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

23	-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]

24	-I Do not perform INDEL calling

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

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

27	-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]

11. 使用bcftools

bcftools和samtools类似，用于处理vcf(variant call format)文件和bcf(binary call format)文件。前者为文本文件，后者为其二进制文件。

bcftools使用简单，最主要的命令是view命令，其次还有index和cat等命令。index和cat命令和samtools中类似。此处主讲使用view命令来进行SNP和Indel calling。该命令的使用方法和例子为：

1	$ bcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s listSample]

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

3	[-1 nGroup1] [-d minFrac] [-U nPerm] [-X permThres]

4	[-T trioType] in.bcf [region]

5

6	$ bcftools view -cvNg abc.bcf > snp_indel.vcf

生成的结果文件为vcf格式，有10列，分别是：

1 参考序列名；

2 variant所在的left-most位置；

3 variant的ID（默认未设置，用’.'表示)；

4 参考序列的allele；

5 variant的allele(有多个alleles，则用’,'分隔);

6 variant/reference QUALity;

7 FILTers applied;

8 variant的信息，使用分号隔开；

9 FORMAT of the genotype fields, separated by colon (optional)；

10 SAMPLE genotypes and per-sample information (optional)。

例如：

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

第8列中显示了对variants的信息描述，比较重要，其中的 Tag 的描述如下：

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

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

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

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

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

10	QCHI2 int   Phred-scaled PCHI2

11	RP int   # permutations yielding a smaller PCHI2

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

13	UGT   string   Most probable genotype configuration without the trio constraint

14	CGT   string   Most probable configuration with the trio constraint

bcftools view 的具体参数如下：

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

14	Consensus/Variant Calling Options:

15	-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)

使用bcftools得到variant calling结果后。需要对结果再次进行过滤。主要依据比对结果中第8列信息。其中的 DP4 一行尤为重要，提供了4个数据：1 比对结果和正链一致的reads数、2 比对结果和负链一致的reads数、3 比对结果在正链的variant上的reads数、4 比对结果在负链的variant上的reads数。可以设定 （value3 + value4）大于某一阈值，才算是variant。比如：

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

12. samtools rmdup

NGS上机测序前需要进行PCR一步，使一个模板扩增出一簇，从而在上机测序的时候表现出为1个点，即一个reads。若一个模板扩增出了多簇，结果得到了多个reads，这些reads的坐标(coordinates)是相近的。在进行了reads比对后需要将这些由PCR duplicates获得的reads去掉，并只保留最高比对质量的read。使用rmdup命令即可完成.

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

本文来自：http://www.chenlianfu.com/?p=1399

网址如下：http://samtools.sourceforge.net/samtools.shtml

Manual Reference Pages  - 

NAME

samtools - Utilities for the Sequence Alignment/Map (SAM) format

bcftools - Utilities for the Binary Call Format (BCF) and VCF

CONTENTS

Synopsis

Description

Samtools Commands And Options

Bcftools Commands And Options

Sam Format

Vcf Format

Examples

Limitations

Author

See Also

SYNOPSIS(大纲）：这个大纲其实详细的说明了运行的命令，如果没有特殊要求就可以直接采用了。下面的东西都是针对这个的描述。

samtools view -bt ref_list.txt -o aln.bam aln.sam.gz

samtools sort aln.bam aln.sorted  ：这个是sort的命令，需要的是aln.bam时你要sort的文件，后面跟的是你可以自己命名的最好和前面保持一致

samtools index aln.sorted.bam

：sort以后要用建立一个索引文件就直接用这个命令

samtools idxstats aln.sorted.bam

samtools view aln.sorted.bam chr2:20,100,000-20,200,000

samtools merge out.bam in1.bam in2.bam in3.bam

samtools faidx ref.fasta

samtools pileup -vcf ref.fasta aln.sorted.bam

samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam

：我们再上面用过的最后snp的提取里

samtools tview aln.sorted.bam ref.fasta

bcftools index in.bcf

bcftools view in.bcf chr2:100-200 > out.vcf

bcftools view -vc in.bcf > out.vcf 2> out.afs

DESCRIPTION

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.

SAMTOOLS COMMANDS AND OPTIONS

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]

BCFTOOLS COMMANDS AND OPTIONS

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.

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:

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

Each bit in the FLAG field is defined as:

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

where the second column gives the string representation of the FLAG field.

VCF FORMAT

The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:

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)

The following table gives the INFO tags used by samtools and bcftools.

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

EXAMPLES

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\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 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.

LIMITATIONS

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.

AUTHOR

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.

SEE ALSO

Samtools website: <http://samtools.sourceforge.net>

samtools-0.1.17

samtools (1)

05 July 2011

上面的内容参考了一下网址的内容

http://www.plob.org/2012/02/04/1703.html

http://www.plob.org/2012/02/04/1700.html

http://www.hudsonalpha.org/gsl/software/bam2fastq.php

http://liucheng.name/670/

http://samtools.sourceforge.net/samtools.shtml

http://blog.sina.com.cn/s/blog_4af3f0d20100xvq1.html

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