文章目录

下载安装
测试数据
命令详解

dict
faidx
index
reheader
rmdup
cat
merge
mpileup

查看参数
mpileup生成的结果
有参考序列的pileup使用
生成一个简单的vcf文件

sort

查看用法
主要参数释义：

split
fastq
fasta
bedcov
depth
flagstat
idxstats
stats
flags
tview
view

bam文件转换为sam文件
sam文件转换为bam文件
比对到反向互补链的reads
比对到正向链的reads
统计共有多少条reads（pair-end-reads这里算一条）参与了比对参考基因组
筛选出比对失败的reads，看序列特征
筛选出比对质量值大于30的情况
筛选出比对成功，但是并不是完全匹配的序列

下载安装


curl -OL https://sourceforge.net/projects/samtools/files/samtools/1.6/samtools-1.6.tar.bz2

tar jxvf samtools-1.6.tar.bz2
cd samtools-1.6
./configure 
make

mv samtools-1.6 samtools
ln -s ~/local/app/samtools/samtools ~/bin/samtools
or 
echo 'export PATH=/home/chengquan/local/app/samtools:$PATH' >> ~/.bashrc
source ~/.bashrc

检查一下是否可以用

[sunchengquan 14:35:50 ~]
$ samtools

Program: samtools (Tools for alignments in the SAM format)
Version: 1.6 (using htslib 1.6)

Usage:   samtools <command> [options]

Commands:
  -- Indexing
     dict           create a sequence dictionary file
     faidx          index/extract FASTA
     index          index alignment

  -- Editing
     calmd          recalculate MD/NM tags and '=' bases
     fixmate        fix mate information
     reheader       replace BAM header
     rmdup          remove PCR duplicates
     targetcut      cut fosmid regions (for fosmid pool only)
     addreplacerg   adds or replaces RG tags
     markdup        mark duplicates

  -- File operations
     collate        shuffle and group alignments by name
     cat            concatenate BAMs
     merge          merge sorted alignments
     mpileup        multi-way pileup
     sort           sort alignment file
     split          splits a file by read group
     quickcheck     quickly check if SAM/BAM/CRAM file appears intact
     fastq          converts a BAM to a FASTQ
     fasta          converts a BAM to a FASTA

  -- Statistics
     bedcov         read depth per BED region
     depth          compute the depth
     flagstat       simple stats
     idxstats       BAM index stats
     phase          phase heterozygotes
     stats          generate stats (former bamcheck)

  -- Viewing
     flags          explain BAM flags
     tview          text alignment viewer
     view           SAM<->BAM<->CRAM conversion
     depad          convert padded BAM to unpadded BAM

以下的命令讲解就是按照这个顺序

测试数据

参考:生物信息数据格式：sam,bam格式

命令详解

官方说明文档：http://www.htslib.org/doc/samtools.html

dict

作用：建立参考基因组字典

[sunchengquan 15:12:43 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reference]
$ samtools dict lambda_virus.fa > lambda_virus.dict

faidx

功能：对fasta格式的文件建立索引，后缀名.fai。根据索引文件和序列文件，可以快速提取任意区域的序列文件。

fasta序列格式要求：每条序列，除了最后一行外，其他行的长度必须相同！

为了方便演示，我们使用bowtie2软件中的测试数据，在example/reference下

查看建立fasta索引

[sunchengquan 14:44:59 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reference]
$ ll
总用量 56K
-rw-r--r-- 1 sunchengquan sunchengquan 49K 7月  27 00:43 lambda_virus.fa

[sunchengquan 14:47:21 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reference]
$ samtools faidx lambda_virus.fa 
[sunchengquan 14:48:32 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reference]
$ ll
总用量 60K
-rw-r--r-- 1 sunchengquan sunchengquan 49K 7月  27 00:43 lambda_virus.fa
-rw-r----- 1 sunchengquan sunchengquan  43 12月 20 14:48 lambda_virus.fa.fai

查看lambda_virus.fa.fai

[sunchengquan 14:49:27 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reference]
$ less -S lambda_virus.fa.fai

gi|9626243|ref|NC_001416.1|     48502   74      70      71

一共5列，每列之间tab分割

第一列：序列的名称
第二列：序列长度
第三列：第一个碱基的偏移量，从0开始计数
第四列：除了最后一行外，序列中每行的碱基数
第五列：除了最后一行外，序列中每行的长度（包括换行符）

提取fasta序列

因为文件中只有一个序列名称，所以我们提取这个序列100-200bp的区间：

[sunchengquan 15:01:01 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reference]
$ samtools faidx lambda_virus.fa 'gi|9626243|ref|NC_001416.1|':100-200 > NC_001416_100_200.fa

index

主要功能：对bam文件建立索引，但在此之前必须进行排序（sort），生成后缀是.bai的文件。

[sunchengquan 15:06:57 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools index tmp.sorted.bam

reheader

作用：替换bam文件的信息

[sunchengquan 15:21:38 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -H tmp.sorted.bam |grep -v '^@HD' > in.header.bam
[sunchengquan 15:21:56 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ less in.header.bam

[sunchengquan 15:31:10 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools reheader -P in.header.bam tmp.sorted.bam > tmp.sorted.h.bam

查看tmp.sorted.h.bam头部信息

[sunchengquan 15:31:20 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -H tmp.sorted.h.bam 
@SQ	SN:gi|9626243|ref|NC_001416.1|	LN:48502
@PG	ID:bowtie2	PN:bowtie2	VN:2.3.4.3	CL:"/home/sunchengquan/local/app/bowtie2-2.3.4.3-linux-x86_64/bowtie2-align-s --wrapper basic-0 -x ../index/lambda_virus -1 reads_1.fq -2 reads_2.fq"

不加-P 参数


[sunchengquan 15:33:06 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools reheader in.header.bam tmp.sorted.bam > tmp.sorted.h.bam
[sunchengquan 15:34:36 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -H tmp.sorted.h.bam 
@SQ	SN:gi|9626243|ref|NC_001416.1|	LN:48502
@PG	ID:bowtie2	PN:bowtie2	VN:2.3.4.3	CL:"/home/sunchengquan/local/app/bowtie2-2.3.4.3-linux-x86_64/bowtie2-align-s --wrapper basic-0 -x ../index/lambda_virus -1 reads_1.fq -2 reads_2.fq"
@PG	ID:samtools	PN:samtools	PP:bowtie2	VN:1.6	CL:samtools reheader in.header.bam tmp.sorted.bam

rmdup

作用：将由PCR duplicates 获得的reads去掉，并保留高比对质量的reads

picard,GATK中也有相似的功能

[sunchengquan 15:36:06 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools rmdup

Usage:  samtools rmdup [-sS] <input.srt.bam> <output.bam>

Option: -s    rmdup for SE reads
        -S    treat PE reads as SE in rmdup (force -s)
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

只需要加-s参数，就可以对单端测序进行去除PCR重复。其实对单端测序去除PCR重复很简单的_{，因为比对flag情况只有0,4,16，只需要它们比对到染色体的起始终止坐标一致即可，flag很容易一致。但是对于双端测序就有点复杂了}

在双端测序里面一大堆的flag情况，双端测序数据，用samtools rmdup效果就很差，所以很多人建议用picard工具的MarkDuplicates 功能~~~

cat

作用：将多个bam文件合并为一个bam文件（merge命令的区别是cat不需要将bam文件提前进行排序）

[sunchengquan 15:50:28 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools cat
Usage: samtools cat [options] <in1.bam>  [... <inN.bam>]
       samtools cat [options] <in1.cram> [... <inN.cram>]

Concatenate BAM or CRAM files, first those in <bamlist.fofn>, then those
on the command line.

Options: -b FILE  list of input BAM/CRAM file names, one per line
         -h FILE  copy the header from FILE [default is 1st input file]
         -o FILE  output BAM/CRAM

merge

功能：合并多个已经sort的bam文件

当有多个样本的bam文件时，可以使用samtools的merge命令将这些bam文件合并为一个排序的且保持所有输入记录并保持现有排序顺序的bam文件

格式：

samtools merge [-nur1f] [-h inh.sam] [-R reg] [-b <list>] <out.bam> <in1.bam> [<in2.bam><in3.bam>…<inN.bam]

-l 指定压缩等级；
-b FILE 输入文件列表，一个文件一行；
-f 强制覆盖同名输出文件；
-h FILE 指定FILE内的’@’头复制到输出bam文件中并替换输出文件的文件头。否则，输出文件的文件头将从第一个输入文件复制过来；
-n 设定输入比对文件是以read名进行排序的而不是以染色体坐标排序的；
-R STRING 合并输入文件的指定区域；
-r 使RG标签添加到每一个比对文件上，标签值来自文件名；
-u 输出的bam文件不压缩；
-c 当多个输入文件包含相同的@RG头ID时，只保留第一个到合并后输出的文件。当合并多个相同样本的不同文件时，非常有用。
-p 与-c参数类似，对于要合并的每一个文件中的@PG ID只保留第一个文件中的@PG。

#-f 参数
[sunchengquan 18:47:33 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools merge tmp.merge.bam tmp.sorted.bam1 tmp.sorted.bam2
[bam_merge] File 'tmp.merge.bam' exists. Please apply '-f' to overwrite. Abort.

[sunchengquan 18:48:53 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools merge -f tmp.merge.bam tmp.sorted.bam1 tmp.sorted.bam2
[sunchengquan 18:49:44 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -H tmp.merge.bam
@HD	VN:1.0	SO:coordinate
@SQ	SN:gi|9626243|ref|NC_001416.1|	LN:48502
@PG	ID:bowtie2	PN:bowtie2	VN:2.3.4.3	CL:"/home/sunchengquan/local/app/bowtie2-2.3.4.3-linux-x86_64/bowtie2-align-s --wrapper basic-0 -x ../index/lambda_virus -1 reads_1.fq -2 reads_2.fq"
@PG	ID:bowtie2-1CE8AF82	PN:bowtie2	VN:2.3.4.3	CL:"/home/sunchengquan/local/app/bowtie2-2.3.4.3-linux-x86_64/bowtie2-align-s --wrapper basic-0 -x ../index/lambda_virus -1 reads_1.fq -2 reads_2.fq"

#-p 参数
[sunchengquan 18:50:04 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools merge -f -p tmp.merge.bam tmp.sorted.bam1 tmp.sorted.bam2
[sunchengquan 18:51:45 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -H tmp.merge.bam
@HD	VN:1.0	SO:coordinate
@SQ	SN:gi|9626243|ref|NC_001416.1|	LN:48502
@PG	ID:bowtie2	PN:bowtie2	VN:2.3.4.3	CL:"/home/sunchengquan/local/app/bowtie2-2.3.4.3-linux-x86_64/bowtie2-align-s --wrapper basic-0 -x ../index/lambda_virus -1 reads_1.fq -2 reads_2.fq"

#-h 参数
#header中多了一行@CO SUNCHENGQUAN 
[sunchengquan 18:51:49 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ cat inh.bam 
@HD	VN:1.0	SO:coordinate
@SQ	SN:gi|9626243|ref|NC_001416.1|	LN:48502
@PG	ID:bowtie2	PN:bowtie2	CL:"/home/sunchengquan/local/app/bowtie2-2.3.4.3-linux-x86_64/bowtie2-align-s --wrapper basic-0 -x ../index/lambda_virus -1 reads_1.fq -2 reads_2.fq"	VN:2.3.4.3
@CO SUNCHENGQUAN 
[sunchengquan 18:52:54 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools merge -h inh.bam -f -p tmp.merge.bam tmp.sorted.bam1 tmp.sorted.bam2
[sunchengquan 18:53:22 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -H tmp.merge.bam
@HD	VN:1.0	SO:coordinate
@SQ	SN:gi|9626243|ref|NC_001416.1|	LN:48502
@PG	ID:bowtie2	PN:bowtie2	CL:"/home/sunchengquan/local/app/bowtie2-2.3.4.3-linux-x86_64/bowtie2-align-s --wrapper basic-0 -x ../index/lambda_virus -1 reads_1.fq -2 reads_2.fq"	VN:2.3.4.3
@CO SUNCHENGQUAN 

#-r 参数
[sunchengquan 18:58:44 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools merge -h inh.bam -f -p -r tmp.merge.bam tmp.sorted.bam1 tmp.sorted.bam2
[sunchengquan 18:58:56 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view tmp.merge.bam |head -1
r523	165	gi|9626243|ref|NC_001416.1|	1	0	*	=	1	0	TTTCGCACTCAATCCGCCGGGCGCGGGGGCGGCGACCTCGCGGGTTTTCGCTATTTAT	(?,8F5CH3+1H<H&.7;F+H?@>%$./?%>F.+/9(H3?4%.=<6A>&"5/.FG?-%	YT:Z:UP	RG:Z:tmp.sorted.bam1
[sunchengquan 18:59:03 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view tmp.merge.bam |tail -1
r9929	141	*	0	0	*	*	0	0	ACCGTAACAAGCAACAGGCAGNCGTGACAGCCAGCAAACCAAAACTCGACCTGACAAACACAGACTGGATTTACGGGGTGGATCTATGAAAATCATC	74)E*11'-G;2G'(!+A26+.E>F'5+.F=@A,/9105>;'E)#F#8C:;G6)G148+<E;)!-B8"#$:7%=?#/H'-F("G4'D/&'&(;2$$+	YT:Z:UP	RG:Z:tmp.sorted.bam2


#-R 参数
[sunchengquan 18:59:12 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools merge -h inh.bam -f -p -r -R 'gi|9626243|ref|NC_001416.1|':1-10 tmp.merge.bam tmp.sorted.bam1 tmp.sorted.bam2
[sunchengquan 19:02:45 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view tmp.merge.bam |wc -l
22
[sunchengquan 19:03:00 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view tmp.merge.bam |awk '{print $4}'
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
4
4
8
8

mpileup

作用：对参考基因组每个位点做碱基堆积，用于call SNP和INDEL。主要是生成BCF、VCF文件或者pileup一个或多个bam文件。比对记录以在@RG中的样本名作为区分标识符。如果样本标识符缺失，那么每一个输入文件则视为一个样本。

mpileup命令中参数比较多，这里只介绍一些常用的参数。

参数	解释
-A	在检测变异中，不忽略异常的reads对
-C	用于调节比对质量的系数，如果reads中含有过多的错配，不能设置为零
-D	输出每个样本的reads深度
-l	BED文件或者包含区域位点的位置列表文件，注意：位置文件包含两列，染色体和位置，从1开始计数。BED文件至少包含3列，染色体、起始和终止位置，开始端从0开始计数。
-r	在指定区域产生pileup，需已建立索引的bam文件，通常和-l参数一起使用
-o/v/g	输出文件类型（标准格式文件或者VCF、BCF文件）
-t	设置FORMAT和INFO的列表内容，以逗号分割
-u	生成未压缩的VCF和BCF文件
-I	跳过INDEL检测
-m	候选INDEL的最小间隔的reads
-f	输入有索引文件的fasta参考序列
-F	含有间隔reads的最小片段
-Q	最低的碱基质量，默认设置是13
-q	最低的mapping质量，默认设置是0

查看参数

samtools mpileup

mpileup生成的结果

[sunchengquan 19:04:21 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools mpileup tmp.sorted.bam |head 
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000
gi|9626243|ref|NC_001416.1|	1	N	0	*	*
gi|9626243|ref|NC_001416.1|	2	N	0	*	*
gi|9626243|ref|NC_001416.1|	3	N	0	*	*
gi|9626243|ref|NC_001416.1|	4	N	0	*	*
gi|9626243|ref|NC_001416.1|	5	N	1	G	/
gi|9626243|ref|NC_001416.1|	6	N	0	*	*
gi|9626243|ref|NC_001416.1|	7	N	1	C	=
gi|9626243|ref|NC_001416.1|	8	N	0	*	*
gi|9626243|ref|NC_001416.1|	9	N	0	*	*
gi|9626243|ref|NC_001416.1|	10	N	1	C	E

在pileup格式中(没有-u或者-g参数)，每一行代表

mpileup生成的结果包含6列

参考序列名
位置
参考碱基
比对上的reads数
比对情况
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；
9 正则式’-[0-9]+[ACGTNacgtn]+’代表在该位点后缺失的碱基；
比对上的碱基的质量

有参考序列的pileup使用

[sunchengquan 19:30:08 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools mpileup -f ../reference/lambda_virus.fa tmp.sorted.bam |tail
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000
gi|9626243|ref|NC_001416.1|	48493	A	3	,,,$	43D
gi|9626243|ref|NC_001416.1|	48494	C	3	,,,	6A<
gi|9626243|ref|NC_001416.1|	48495	A	2	,$,	/>
gi|9626243|ref|NC_001416.1|	48496	G	2	,,	?D
gi|9626243|ref|NC_001416.1|	48497	G	1	,	<
gi|9626243|ref|NC_001416.1|	48498	T	0	*	*
gi|9626243|ref|NC_001416.1|	48499	T	1	,$	B
gi|9626243|ref|NC_001416.1|	48500	A	0	*	*
gi|9626243|ref|NC_001416.1|	48501	C	0	*	*
gi|9626243|ref|NC_001416.1|	48502	G	0	*	*

生成一个简单的vcf文件

[sunchengquan 19:38:45 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools mpileup -uvf ../reference/lambda_virus.fa tmp.sorted.bam |head -25
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000
##fileformat=VCFv4.2
##FILTER=<ID=PASS,Description="All filters passed">
##samtoolsVersion=1.6+htslib-1.6
##samtoolsCommand=samtools mpileup -uvf ../reference/lambda_virus.fa tmp.sorted.bam
##reference=file://../reference/lambda_virus.fa
##contig=<ID=gi|9626243|ref|NC_001416.1|,length=48502>
##ALT=<ID=*,Description="Represents allele(s) other than observed.">
##INFO=<ID=INDEL,Number=0,Type=Flag,Description="Indicates that the variant is an INDEL.">
##INFO=<ID=IDV,Number=1,Type=Integer,Description="Maximum number of reads supporting an indel">
##INFO=<ID=IMF,Number=1,Type=Float,Description="Maximum fraction of reads supporting an indel">
##INFO=<ID=DP,Number=1,Type=Integer,Description="Raw read depth">
##INFO=<ID=VDB,Number=1,Type=Float,Description="Variant Distance Bias for filtering splice-site artefacts in RNA-seq data (bigger is better)",Version="3">
##INFO=<ID=RPB,Number=1,Type=Float,Description="Mann-Whitney U test of Read Position Bias (bigger is better)">
##INFO=<ID=MQB,Number=1,Type=Float,Description="Mann-Whitney U test of Mapping Quality Bias (bigger is better)">
##INFO=<ID=BQB,Number=1,Type=Float,Description="Mann-Whitney U test of Base Quality Bias (bigger is better)">
##INFO=<ID=MQSB,Number=1,Type=Float,Description="Mann-Whitney U test of Mapping Quality vs Strand Bias (bigger is better)">
##INFO=<ID=SGB,Number=1,Type=Float,Description="Segregation based metric.">
##INFO=<ID=MQ0F,Number=1,Type=Float,Description="Fraction of MQ0 reads (smaller is better)">
##INFO=<ID=I16,Number=16,Type=Float,Description="Auxiliary tag used for calling, see description of bcf_callret1_t in bam2bcf.h">
##INFO=<ID=QS,Number=R,Type=Float,Description="Auxiliary tag used for calling">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description="List of Phred-scaled genotype likelihoods">
#CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	tmp.sorted.bam
gi|9626243|ref|NC_001416.1|	1	.	G	<*>	0	.	DP=1;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0	PL	0,0,0
gi|9626243|ref|NC_001416.1|	2	.	G	<*>	0	.	DP=2;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0	PL	0,0,0
gi|9626243|ref|NC_001416.1|	2	.	GGCG	GGCGCGGGGGCG	0	.	INDEL;IDV=1;IMF=0.5;DP=2;I16=0,0,1,0,0,0,41,1681,0,0,42,1764,0,0,0,0;QS=0,1;SGB=-0.379885;MQ0F=0	PL	41,3,0

sort

主要功能：对bam文件进行排序（不能对sam文件进行排序）

查看用法

[sunchengquan 19:45:09 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools sort
Usage: samtools sort [options...] [in.bam]
Options:
  -l INT     Set compression level, from 0 (uncompressed) to 9 (best)
  -m INT     Set maximum memory per thread; suffix K/M/G recognized [768M]
  -n         Sort by read name
  -t TAG     Sort by value of TAG. Uses position as secondary index (or read name if -n is set)
  -o FILE    Write final output to FILE rather than standard output
  -T PREFIX  Write temporary files to PREFIX.nnnn.bam
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -O, --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]
  -@, --threads INT
               Number of additional threads to use [0]

主要参数释义：

-l：设置文件压缩等级，0不压缩，9压缩最高
-m：每个线程运行内存大小（可使用K M G表示）
-n：按照read名称进行排序
-o：排序后的输出文件
-T：PREFIX临时文件前缀
-@：设置排序和压缩的线程数，默认单线程

[sunchengquan 22:24:41 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools sort  -l 9 -m 90M -n -o tmp.sorted.bam -T sorted -@ 2 tmp.bam
[bam_sort_core] merging from 0 files and 2 in-memory blocks...

查看tmp.sorted.bam

[sunchengquan 22:35:28 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view tmp.sorted.bam|less -S


r1      99      gi|9626243|ref|NC_001416.1|     18401   42      122M    =       18430
r1      147     gi|9626243|ref|NC_001416.1|     18430   42      198M    =       18401
r2      99      gi|9626243|ref|NC_001416.1|     8886    42      275M    =       8973
r2      147     gi|9626243|ref|NC_001416.1|     8973    42      188M    =       8886
r3      83      gi|9626243|ref|NC_001416.1|     11599   42      338M    =       11596
r3      163     gi|9626243|ref|NC_001416.1|     11596   42      67M     =       11599
r4      99      gi|9626243|ref|NC_001416.1|     40075   42      184M    =       40211
r4      147     gi|9626243|ref|NC_001416.1|     40211   42      48M     =       40075

不加-n 参数

[sunchengquan 22:36:58 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools sort  -l 9 -m 90M  -o tmp.sorted.bam -T sorted -@ 2 tmp.bam
[bam_sort_core] merging from 0 files and 2 in-memory blocks...
[sunchengquan 22:38:10 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view tmp.sorted.bam|less -S


r523    165     gi|9626243|ref|NC_001416.1|     1       0       *       =       1
r2543   165     gi|9626243|ref|NC_001416.1|     1       0       *       =       1
r4819   163     gi|9626243|ref|NC_001416.1|     1       24      4M16I183M       =
r523    89      gi|9626243|ref|NC_001416.1|     1       0       3M26I196M       =
r2543   89      gi|9626243|ref|NC_001416.1|     1       42      214M    =       1
r6080   177     gi|9626243|ref|NC_001416.1|     1       42      4M2I54M =       5850
r9207   177     gi|9626243|ref|NC_001416.1|     1       42      2M6I204M        =
r9555   113     gi|9626243|ref|NC_001416.1|     1       42      116M    =       5826
r3903   99      gi|9626243|ref|NC_001416.1|     2       42      1M8I238M        =
r874    99      gi|9626243|ref|NC_001416.1|     4       42      49M     =       232
r3796   113     gi|9626243|ref|NC_001416.1|     8       42      120M    =       5912

split

作用：根据read group 分割bam文件


[sunchengquan 17:04:02 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ find *|grep '[0-9]$' |awk '{print "@RG\tID:"$0}' >> inh.bam 
[sunchengquan 17:04:06 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools reheader -P inh.bam tmp.merge.bam > tmp.merge.h.bam
[sunchengquan 17:04:14 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -H tmp.merge.h.bam 
@HD	VN:1.0	SO:coordinate
@SQ	SN:gi|9626243|ref|NC_001416.1|	LN:48502
@PG	ID:bowtie2	PN:bowtie2	CL:"/home/sunchengquan/local/app/bowtie2-2.3.4.3-linux-x86_64/bowtie2-align-s --wrapper basic-0 -x ../index/lambda_virus -1 reads_1.fq -2 reads_2.fq"	VN:2.3.4.3
@RG	ID:tmp.sorted.bam1
@RG	ID:tmp.sorted.bam2
[sunchengquan 17:04:27 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools split tmp.merge.h.bam
[sunchengquan 17:04:43 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ ll * |grep '.*merge'
-rw-r----- 1 sunchengquan sunchengquan 2.7M 12月 21 16:29 tmp.merge.bam
-rw-r----- 1 sunchengquan sunchengquan 2.5M 12月 21 17:04 tmp.merge.h_0.bam
-rw-r----- 1 sunchengquan sunchengquan 2.5M 12月 21 17:04 tmp.merge.h_1.bam
-rw-r----- 1 sunchengquan sunchengquan 2.7M 12月 21 17:04 tmp.merge.h.bam

fastq

作用：bam文件转换为fastq


[sunchengquan 17:11:19 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools fastq tmp.sorted.bam -1 read1.fq.gz -2 read2.fq.gz 
[M::bam2fq_mainloop] discarded 0 singletons
[M::bam2fq_mainloop] processed 20000 reads
[sunchengquan 17:13:44 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ ll *|grep 'gz$'
-rw-r----- 1 sunchengquan sunchengquan 1.1M 12月 21 17:13 read1.fq.gz
-rw-r----- 1 sunchengquan sunchengquan 1.1M 12月 21 17:13 read2.fq.gz
[sunchengquan 17:14:20 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ zcat read1.fq.gz |wc -l 
40000
[sunchengquan 17:14:58 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ cat read_1.fq |wc -l 
cat: read_1.fq: 没有那个文件或目录
0
[sunchengquan 17:15:19 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ cat reads_1.fq |wc -l 
40000

fasta

bam文件转换为fasta


[sunchengquan 17:15:31 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools fasta tmp.sorted.bam -1 read1.fa.gz -2 read2.fa.gz 
[M::bam2fq_mainloop] discarded 0 singletons
[M::bam2fq_mainloop] processed 20000 reads
[sunchengquan 17:18:56 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ zcat read1.fa.gz |wc -l 
20000
[sunchengquan 17:19:09 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ zcat read1.fa.gz |head
>r523
GACTTCCATTGTTCATTCCACGGACACAAACAGAGAAAGGAAACGACAGAGNCCAAAAAGCCTNGCTTTCAGCACCTGTCGTTTCCTTTCTTTTCAGAGGGTATTTTAAATAAAAACATTAAGTTATGACGAAGAAGAACGGAAACGCCTTAAACCGGAAAATTTTCATAAATAGCGAAAACCCGCGAGGNCGCCGCCCCCGCGCCCGGCGGANTGAGTGCGAAA
>r2543
CAGCTGCTTTTTGTTGACTTCCATTGTTCATTCCACGGACAAAAACAGAGANAGGAAACGACAGAGGCCAAAAAGCCTCGCTTTCAGCACCTGTCGTTTCCTTTCTTTTCAGAGGGTATTTTAAATAAAAACATTAAGTTATGACGAAGAAGAACGGAAACGCCTTAAACCGGAAAAATTTCNTAAATAGCGAAAACCCGCGAGGTCGCCGCCC
>r9555
TCCCTTCTTTTAAGAGGGTATTTTAAATAAAAACATTAAGTTATGACGAAGAAGAACGGAAACGCCTTAAACCGGAAAATTTTCATAAATAGCGAAAACCCGCGAGGTCGCCGCCC
>r3903
NGCGCGGGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGTAAGCGAGGCTTTTTGGCCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCAACAAAAAGCAGCTGGCTGACATTTTCGGTGCGAGTATCCG
>r874
CGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTTCGGTTTAAGG

bedcov

输出每个panel的总的碱基数；或者将一个panel中每个碱基的深度加到一起。panel指的是每个bed中的一个窗口，由chrID，start，end定义使用方法

depth

输出每个碱基的深度，即匹配到的次数。可以通过-b添加bed文件，来缩小范围。另外。默认最大深度为8000，如果是高深度测序应该通过-d或-m来定义更大的深度。如果需要统计深度为0的位点，应该加上-a选项。参数较多，主要参数如下：

-a：输出所有的碱基深度（包括0）
-b/-r：控制深度的范围（后面跟染色体）
-f：bam文件名字
-l：设置read长度阈值
-d/-m：最大覆盖深度
-q：碱基质量阈值
-Q：比对质量阈值

查看100-200bp区域的深度


[sunchengquan 19:14:33 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools index tmp.sorted.bam
[sunchengquan 19:15:19 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools depth -r 'gi|9626243|ref|NC_001416.1|':100-200 tmp.sorted.bam > tmp.sorted.bam.depth
[sunchengquan 19:15:22 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ cat tmp.sorted.bam.depth|head
gi|9626243|ref|NC_001416.1|	100	30
gi|9626243|ref|NC_001416.1|	101	31
gi|9626243|ref|NC_001416.1|	102	31
gi|9626243|ref|NC_001416.1|	103	31
gi|9626243|ref|NC_001416.1|	104	30
gi|9626243|ref|NC_001416.1|	105	30
gi|9626243|ref|NC_001416.1|	106	30
gi|9626243|ref|NC_001416.1|	107	30
gi|9626243|ref|NC_001416.1|	108	30
gi|9626243|ref|NC_001416.1|	109	29

结果是tab分割的三列

第一列：染色体名称

第二列：碱基位置

第三列：测序深度

flagstat

对bam文件进行简单的统计，一共有13项，根据QC结果，每一项都分PASS和FAIL，一般都是PASS，毕竟一般QC都在比对前做过了，低质量的大都过滤了

[sunchengquan 19:15:49 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools flagstat tmp.sorted.bam
20000 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
18995 + 0 mapped (94.98% : N/A)
20000 + 0 paired in sequencing
10000 + 0 read1
10000 + 0 read2
18332 + 0 properly paired (91.66% : N/A)
18416 + 0 with itself and mate mapped
579 + 0 singletons (2.90% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

总的reads

20000 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates

总体reads比对率

18995 + 0 mapped (94.98% : N/A)

PEreads

20000 + 0 paired in sequencing

read1

10000 + 0 read1

read2

10000 + 0 read2

PE reads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

18332 + 0 properly paired (91.66% : N/A)

PE中两条都比对到参考序列上的reads数

18416 + 0 with itself and mate mapped

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

579 + 0 singletons (2.90% : N/A)

PE中两条分别比对到两条不同的参考序列的reads数

0 + 0 with mate mapped to a different chr

PE中两条分别比对到两条不同的参考序列的reads数（比对质量>=5）

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

idxstats

对bam/sam/cram文件的索引进行输出，一共四列：

chrID 序列名
chrLength 序列长度
mapped reads 比对上的reads数
unmapped reads 未必对数目

文件的索引文件必须存在于输入文件相同目录下，如果没有需要先建立索引

[sunchengquan 19:49:46 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools idxstats tmp.sorted.bam
gi|9626243|ref|NC_001416.1|	48502	18995	579
*	0	0	426

stats

对bam文件做详细统计,其统计结果可用mics/plot-bamstats作图

SN Summary Number
FFQ First Fragment Qualities
LFQ Last Fragment Quailites
GCF GC Content of first fragments
GCL GC Content of last fragments
GCC ACGT content per cycle
IS Insert sizes
RL Read lengths
ID Indel distribution
IC Indels per cycle
COV Coverage distribution
GCD GC-depth

提取SN

[sunchengquan 20:08:45 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools stats tmp.sorted.bam|grep '^SN' |cut -f 2-
raw total sequences:	20000
filtered sequences:	0
sequences:	20000
is sorted:	1
1st fragments:	10000
last fragments:	10000
reads mapped:	18995
reads mapped and paired:	18416	# paired-end technology bit set + both mates mapped
reads unmapped:	1005
reads properly paired:	18332	# proper-pair bit set
reads paired:	20000	# paired-end technology bit set
reads duplicated:	0	# PCR or optical duplicate bit set
reads MQ0:	11	# mapped and MQ=0
reads QC failed:	0
non-primary alignments:	0
total length:	2178385	# ignores clipping
bases mapped:	2079519	# ignores clipping
bases mapped (cigar):	2079519	# more accurate
bases trimmed:	0
bases duplicated:	0
mismatches:	51081	# from NM fields
error rate:	2.456385e-02	# mismatches / bases mapped (cigar)
average length:	108
maximum length:	366
average quality:	16.3
insert size average:	251.6
insert size standard deviation:	44.2
inward oriented pairs:	8485
outward oriented pairs:	0
pairs with other orientation:	40
pairs on different chromosomes:	0

提取IS

[sunchengquan 21:25:35 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools stats tmp.sorted.bam|grep '^IS' |cut -f 2-|tail
357	7	7	0	0
358	5	5	0	0
359	6	6	0	0
360	4	4	0	0
361	7	7	0	0
362	5	5	0	0
363	1	1	0	0
364	0	0	0	0
365	1	1	0	0
366	4	4	0	0

flags

将字符型和数字型flag相互转换

[sunchengquan 21:29:13 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools flags 83
0x53	83	PAIRED,PROPER_PAIR,REVERSE,READ1
[sunchengquan 21:29:20 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools flags read2
0x80	128	READ2
[sunchengquan 21:30:01 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools flags read1
0x40	64	READ1
[sunchengquan 21:30:24 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools flags read1,read2
0xc0	192	READ1,READ2

tview

直观显示reads比对到基因组的情况，和基因组浏览器类似

主要参数：

-d：输出类型
-p：直接定位给定位置
-s：reads显示

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

首先利用sort进行排序后，在利用index建立索引后，用下面命令：

[sunchengquan 21:35:17 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools tview -p 'gi|9626243|ref|NC_001416.1|':300 tmp.sorted.bam

示意图：
xxxx

利用颜色来标注比对质量、碱基质量、核苷酸等。

>=30 的碱基质量或比对质量使用白色表示

20-39的用黄色表示

10-19的用绿色表示

0-9的用蓝色表示

使用字母r切换显示read name等。

具体的操作说明可以？键查看。

view

sam和bam文件之间相互转换，针对bam文件进行相关操作。bam文件是sam文件的二进制格式，占据内存较小且运算速度快

重要参数释义:

-b：输出bam格式，用于后续分析
-C：输出CRAM文件
-1：快速压缩（需要-b）
-u：输出未压缩的bam文件，节约时间，占据较多磁盘空间（需要-b）
-h：默认输出sam文件不带表头，该参数设定后输出带表头信息
-H：仅仅输出表头信息
-c：仅打印匹配数
-o：输出文件（stdout标准输出）
-U：输出没有经过过滤选择的reads
-t：制表分隔符文件（需要提供额外的参考数据，比如参考基因组的索引）
-L：仅包括和bed文件有重叠的reads
-r：仅输出在STR读段组中的reads
-R：仅输出特定reads
-q：定位的质量大于INT[默认0]
-l：仅输出在STR 库中的reads
-F：过滤标识（flag）（去除匹配标识对应的reads，保留剩余的）[默认0]
-f：匹配标识（flag）（保留匹配的标识对应的reads）[默认0]
-T：使用fasta格式的参考序列

bam文件转换为sam文件

[sunchengquan 21:41:12 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -h tmp.sorted.bam >tmp.sam

sam文件转换为bam文件

[sunchengquan 21:41:12 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -bS tmp.sam > tmp.sorted.bam

比对到反向互补链的reads

$ samtools view -f 16 tmp.sorted.bam|head -1
r523	89	gi|9626243|ref|NC_001416.1|	1	0	3M26I196M	=	1	0	TTTCGCACTCANTCCGCCGGGCGCGGGGGCGGCGNCCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCNAGGCTTTTTGGNCTCTGTCGTTTCCTTTCTCTGTTTGTGTCCGTGGAATGAACAATGGAAGTC	"1&;;,*'<-1G$427,3>D:+F+';13<:06=F2$:71D5G><@E@;0>");,G0=@7)!:H9&D.C?=1@00H=81E3C,8@<=B?;9;'.+2A!H<#!073&7=8&9G!CHDCG*>%'1C!;9!.+68,5?9304=12>4:18:C&%G7+H6-8CD0$:2"./(#D5E14?5=230>"?4?>G&@>'60&(?;44:3G5/B::4'9FC);(52!@<<6F5=5	AS:i:-97	XN:i:0	XM:i:7	XO:i:1	XG:i:26	NM:i:33	MD:Z:0G0G0G5A126G11C24T26	YT:Z:UP

比对到正向链的reads

[sunchengquan 21:55:03 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -F 16 tmp.sorted.bam|head -1
r523	165	gi|9626243|ref|NC_001416.1|	1	0	*	=	1	0	TTTCGCACTCAATCCGCCGGGCGCGGGGGCGGCGACCTCGCGGGTTTTCGCTATTTAT	(?,8F5CH3+1H<H&.7;F+H?@>%$./?%>F.+/9(H3?4%.=<6A>&"5/.FG?-%	YT:Z:UP

统计共有多少条reads（pair-end-reads这里算一条）参与了比对参考基因组

[sunchengquan 21:56:24 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -c tmp.sorted.bam
20000
[sunchengquan 21:58:02 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ echo `samtools view -c tmp.sorted.bam`/2 |bc
10000

筛选出比对失败的reads，看序列特征

[sunchengquan 21:59:33 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -cf 4 tmp.sorted.bam
1005

[sunchengquan 22:09:01 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -f 4 tmp.bam|cut -f10 |head -3
GCGTNGTACNNGNNGATGNTTACGACTAANACTTNNNNNTNCNGNNT
ANTGGNTNAGCGGNCGNAGNGNTCNGNAGCNANNAANCTGNTATGNTNNNTNNATCNNNCNNNNGTGNNAANNNCCAGNNNNGGCNNNNCATACANNNTNNNCNTNNNTACANNATANNCGNNATTNNACNGTNNCGNGNNCCGNGTNNNNN
NNTNNNNNAGNTNAANANANGTNTTGNGAANCTNNANACNNCTTTTNNNNTATGNTNNTNAGCCTAG

筛选出比对质量值大于30的情况

[sunchengquan 22:16:19 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -q 30 tmp.bam |awk '{print $1,$5}'|head -3
r1 42
r1 42
r2 42

筛选出比对成功，但是并不是完全匹配的序列

[sunchengquan 22:17:46 ~/local/app/bowtie2-2.3.4.3-linux-x86_64/example/reads]
$ samtools view -F 4 tmp.bam |awk '{print $6}'|grep '[IDNSHPX]'|head -4
72M2D119M
126M3D61M
70M1D71M
53M5D134M

samtools用法详解