1、bwa和samtools的安装

    BWA的安装

1.下载BWA

2.解压

3.编译

Samtool安裝
1.下载Samtool

2.解压Samtool

3.安装

之外Samtool还有一些脚本程序在misc这个文件夹里面

最后将bwa和samtools的工作目录加入PATH

这样就可以直接使用bwa和Samtool了。

samtools一般是统计比对信息的，一些处理工作等。

bwa的使用：

BWA SYNOPSIS语法：

1）bwa index ref.fa

2）bwa aln ref.fa short_read.fq > aln_sa.sai

3）bwa samse ref.fa aln_sa.sai short_read.fq > aln-se.sam

3）bwa sampe ref.fa aln_sa1.sai aln_sa2.sai read1.fq read2.fq > aln-pe.sam

【bwa bwasw ref.fa long_read.fq > aln.sam

 bwa mem ref.fa reads.fq > aln-se.sam

 bwa mem ref.fa read1.fq read2.fq > aln-pe.sam】

bwa的使用需要两种输入文件：

1. Reference genome data（fasta格式 .fa, .fasta, .fna）

2. Short reads data (fastaq格式 .fastaq, .fq)

step 1: 建立 Index
根据reference genome data(e.g. reference.fa) 建立 Index File

bwa index 指令更多的用法及 options，通过以下的命令来查看

step 2: 寻找 SA coordinates
如果是pair-end 数据（leftRead.fastq和rightRead.fastq）两个文件分别处理；single-end数据则直接处理。

如果希望多线程运行，在其中加入 -t这个参数，另外-f这个参数可以指定结果输出文件，如:

step 3:转换SA coordinates输出为sam
如果是pair-end数据

如果是single reads数据

值此Reads的mapping工作已经完成。接下来用samtools后续处理。

3、bowtie的使用

Usage:

 bowtie [options]* <ebwt> {-1 <m1> -2 <m2> | --12 <r> | <s>} [<hit>]

 <m1>    Comma-separated list of files containing upstream mates (or the

         sequences themselves, if -c is set) paired with mates in <m2>

 <m2>    Comma-separated list of files containing downstream mates (or the

         sequences themselves if -c is set) paired with mates in <m1>

 <r>     Comma-separated list of files containing Crossbow-style reads.  Can be

         a mixture of paired and unpaired.  Specify "-" for stdin.

 <s>     Comma-separated list of files containing unpaired reads, or the

         sequences themselves, if -c is set.  Specify "-" for stdin.

 <hit>   File to write hits to (default: stdout)

现在举个例子来探讨bowtie的使用方法：现在有GENOME.fa、高通量测序数据Reads.fa，我们希望将Reads.fa比对到基因组GENOME.fa上。

（一）、对Reference文件(GENOME.fa)建库

建库步骤可能需要1h甚至几个小时，建议在后台执行：
nohup bowtie-build GENOME.fa GENOME.fa &

（二）、将Reads.fa比对到GENOME.fa上，只能比对到正链，且匹配到基因组不多于20个不同位置，允许有1个错配

• 注：

• -f 指定query文件为fasta格式

• -a 保留所有比对结果

• -m 指定最大比对到基因组的次数

• -v 允许最大错配数，为[0-2]

• --al 能map到GENOME的reads，fasta格式

• --un 不能map到GENOME的reads，fasta格式

• --norc 不输出匹配到负链的结果；如果不想输出比对到正链的结果，则用"--nofw"。不指定该选项则正负链结果都输出

• 后面依次写上GENOME索引文件，Reads文件，输出结果文件Reads.bwt，日志文件log。

（三）、bowtie输出结果的说明

• 1. query id

• 2. "+"表示正向match；"-"表示对query作反向互补后match

• 3. reference id

• 4. 第2列为"+"时，表示query 第一个碱基map到reference(5'->3')上的位置，0-based(以0开始)；第2列为"-"时，表示query的反向互补序列第一 个碱基map到reference(5'->3')上的位置，0-based(以0开始)

• 5. 如果第2列为"+"，则和query序列一致；否则，和query序列反向互补

• 6. 质量文件，如果query文件为fasta格式，则无法获取质量文件，用I代替，I的数量与query序列长度一致

• 7. 当前query能map到GENOME的4个不同位置

• 8. 如果存在第8列，表示有mismatch。第8列可以分为三个部分，最左端的数字，中间的碱基为reference碱基，最右端的碱基为query碱基，下面分情况讨论：

• 第2列为"+"时：最左端的数字9表示query从5'端数起，第10个碱基为"T"，而对应的reference为"G";
  第2列为"-"时：最左端的数字9表示query先作反向互补，然后从3'端数起，第10个碱基为"T"，而对应的reference为"G";

4、bowtie2

1）下载（wget http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.3/bowtie2-2.2.3-linux-x86_64.zip/download）

解压   unzip bowtie2-。。。。

2）Set the BT2_HOME environment variable to point to the new Bowtie 2 directory containing the bowtie2, bowtie2-build and bowtie2-inspect binaries. 【将 其加入环境变量（确保bowtie2,bowtie2-build or bowtie2-inspect能使用】

3）建索引：

  $ bowtie2-build genome.fasta index

【【命令： bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r>} [-S <sam>]

 <bt2-idx>  Index filename prefix (minus trailing .X.bt2).

            NOTE: Bowtie 1 and Bowtie 2 indexes are not compatible.

 <m1>       Files with #1 mates, paired with files in <m2>.

            Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).

 <m2>       Files with #2 mates, paired with files in <m1>.

            Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).

 <r>        Files with unpaired reads.

            Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).

 <sam>      File for SAM output (default: stdout)

 <m1>, <m2>, <r> can be comma-separated lists (no whitespace) and can be

 specified many times.  E.g. '-U file1.fq,file2.fq -U file3.fq'.】】

$ bowtie2 -p 8 -x index -1 reads1.fq -2 reads2.fq -S out.sam

以一个例子详细说明：

【【【【Bowtie 2 comes with some example files to get you started. The example files are not scientifically significant; we use theLambda phage reference genome simply because it's short, and the reads were generated by a computer program, not a sequencer. However, these files will let you start running Bowtie 2 and downstream tools right away.

First follow the manual instructions to obtain Bowtie 2. Set the BT2_HOME environment variable to point to the new Bowtie 2 directory containing the bowtie2, bowtie2-build and bowtie2-inspect binaries. This is important, as the BT2_HOME variable is used in the commands below to refer to that directory.

Indexing a reference genome

To create an index for the Lambda phage reference genome included with Bowtie 2, create a new temporary directory (it doesn't matter where), change into that directory, and run:

$BT2_HOME/bowtie2-build $BT2_HOME/example/reference/lambda_virus.fa lambda_virus

The command should print many lines of output then quit. When the command completes, the current directory will contain four new files that all start with lambda_virus and end with .1.bt2, .2.bt2, .3.bt2, .4.bt2, .rev.1.bt2, and .rev.2.bt2. These files constitute the index - you're done!

You can use bowtie2-build to create an index for a set of FASTA files obtained from any source, including sites such asUCSC, NCBI, and Ensembl. When indexing multiple FASTA files, specify all the files using commas to separate file names. For more details on how to create an index with bowtie2-build, see the manual section on index building. You may also want to bypass this process by obtaining a pre-built index. See using a pre-built index below for an example.

Aligning example reads

Stay in the directory created in the previous step, which now contains the lambda_virus index files. Next, run:

$BT2_HOME/bowtie2 -x lambda_virus -U $BT2_HOME/example/reads/reads_1.fq -S eg1.sam

This runs the Bowtie 2 aligner, which aligns a set of unpaired reads to the Lambda phage reference genome using the index generated in the previous step. The alignment results in SAM format are written to the file eg1.sam, and a short alignment summary is written to the console. (Actually, the summary is written to the "standard error" or "stderr" filehandle, which is typically printed to the console.)

To see the first few lines of the SAM output, run:

head eg1.sam

You will see something like this:

@HD VN:1.0  SO:unsorted@SQ SN:gi|9626243|ref|NC_001416.1|  LN:48502@PG ID:bowtie2  PN:bowtie2  VN:2.0.1r1  0   gi|9626243|ref|NC_001416.1| 18401   42  122M    *   0   0   TGAATGCGAACTCCGGGACGCTCAGTAATGTGACGATAGCTGAAAACTGTACGATAAACNGTACGCTGAGGGCAGAAAAAATCGTCGGGGACATTNTAAAGGCGGCGAGCGCGGCTTTTCCG  +"@6<:27(F&5)9)"B:%B+A-%5A?2$HCB0B+0=D<7E/<.03#!.F77@6B==?C"7>;))%;,3-$.A06+<-1/@@?,26">=?*@'0;$:;??G+:#+(A?9+10!8!?()?7C>  AS:i:-5 XN:i:0  XM:i:3  XO:i:0  XG:i:0  NM:i:3  MD:Z:59G13G21G26    YT:Z:UUr2  0   gi|9626243|ref|NC_001416.1| 8886    42  275M    *   0   0   NTTNTGATGCGGGCTTGTGGAGTTCAGCCGATCTGACTTATGTCATTACCTATGAAATGTGAGGACGCTATGCCTGTACCAAATCCTACAATGCCGGTGAAAGGTGCCGGGATCACCCTGTGGGTTTATAAGGGGATCGGTGACCCCTACGCGAATCCGCTTTCAGACGTTGACTGGTCGCGTCTGGCAAAAGTTAAAGACCTGACGCCCGGCGAACTGACCGCTGAGNCCTATGACGACAGCTATCTCGATGATGAAGATGCAGACTGGACTGC (#!!'+!$""%+(+)'%)%!+!(&++)''"#"#&#"!'!("%'""("+&%$%*%%#$%#%#!)*'(#")(($&$'&%+&#%*)*#*%*')(%+!%%*"$%"#+)$&&+)&)*+!"*)!*!("&&"*#+"&"'(%)*("'!$*!!%$&&&$!!&&"(*"$&"#&!$%'%"#)$#+%*+)!&*)+(""#!)!%*#"*)*')&")($+*%%)!*)!('(%""+%"$##"#+(('!*(($*'!"*('"+)&%#&$+('**$$&+*&!#%)')'(+(!%+ AS:i:-14    XN:i:0  XM:i:8  XO:i:0  XG:i:0  NM:i:8  MD:Z:0A0C0G0A108C23G9T81T46 YT:Z:UUr3  16  gi|9626243|ref|NC_001416.1| 11599   42  338M    *   0   0   GGGCGCGTTACTGGGATGATCGTGAAAAGGCCCGTCTTGCGCTTGAAGCCGCCCGAAAGAAGGCTGAGCAGCAGACTCAAGAGGAGAAAAATGCGCAGCAGCGGAGCGATACCGAAGCGTCACGGCTGAAATATACCGAAGAGGCGCAGAAGGCTNACGAACGGCTGCAGACGCCGCTGCAGAAATATACCGCCCGTCAGGAAGAACTGANCAAGGCACNGAAAGACGGGAAAATCCTGCAGGCGGATTACAACACGCTGATGGCGGCGGCGAAAAAGGATTATGAAGCGACGCTGTAAAAGCCGAAACAGTCCAGCGTGAAGGTGTCTGCGGGCGAT  7F$%6=$:9B@/F'>=?!D?@0(:A*)7/>9C>6#1<6:C(.CC;#.;>;2'$4D:?&B!>689?(0(G7+0=@37F)GG=>?958.D2E04C<E,*AD%G0.%$+A:'H;?8<72:88?E6((CF)6DF#.)=>B>D-="C'B080E'5BH"77':"@70#4%A5=6.2/1>;9"&-H6)=$/0;5E:<8G!@::1?2DC7C*;@*#.1C0.D>H/20,!"C-#,6@%<+<D(AG-).?�.00'@)/F8?B!&"170,)>:?<A7#1(A@0E#&A.*DC.E")AH"+.,5,2>5"2?:G,F"D0B8D-6$65D<D!A/38860.*4;4B<*31?6  AS:i:-22    XN:i:0  XM:i:8  XO:i:0  XG:i:0  NM:i:8  MD:Z:80C4C16A52T23G30A8T76A41   YT:Z:UUr4  0   gi|9626243|ref|NC_001416.1| 40075   42  184M    *   0   0   GGGCCAATGCGCTTACTGATGCGGAATTACGCCGTAAGGCCGCAGATGAGCTTGTCCATATGACTGCGAGAATTAACNGTGGTGAGGCGATCCCTGAACCAGTAAAACAACTTCCTGTCATGGGCGGTAGACCTCTAAATCGTGCACAGGCTCTGGCGAAGATCGCAGAAATCAAAGCTAAGT(=8B)GD04*G%&4F,1'A>.C&7=F$,+#6!))43C,5/5+)?-/0>/D3=-,2/+.1?@->;)00!'3!7BH$G)HG+ADC'#-9F)7<7"$?&.>0)@5;4,!0-#C!15CF8&HB+B==H>7,/)C5)5*+(F5A%D,EA<(>G9E0>7&/E?4%;#'92)<5+@7:A.(BG@BG86@.G AS:i:-1 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:77C106 YT:Z:UUr5  0   gi|9626243|ref|NC_001416.1| 48010   42  138M    *   0   0   GTCAGGAAAGTGGTAAAACTGCAACTCAATTACTGCAATGCCCTCGTAATTAAGTGAATTTACAATATCGTCCTGTTCGGAGGGAAGAACGCGGGATGTTCATTCTTCATCACTTTTAATTGATGTATATGCTCTCTT  9''%<D)A03E1-*7=),:F/0!6,D9:H,<9D%:0B(%'E,(8EFG$E89B$27G8F*2+4,-!,0D5()&=(FGG:5;3*@/.0F-G#5#3->('FDFEG?)5.!)"AGADB3?6(@H(:B<>6!>;>6>G,."?%  AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:138    YT:Z:UUr6  16  gi|9626243|ref|NC_001416.1| 41607   42  72M2D119M   *   0   0   TCGATTTGCAAATACCGGAACATCTCGGTAACTGCATATTCTGCATTAAAAAATCAACGCAAAAAATCGGACGCCTGCAAAGATGAGGAGGGATTGCAGCGTGTTTTTAATGAGGTCATCACGGGATNCCATGTGCGTGACGGNCATCGGGAAACGCCAAAGGAGATTATGTACCGAGGAAGAATGTCGCT 1H#G;H"$E*E#&"*)2%66?=9/9'=;4)4/>@%+5#@#$4A*!<D=="8#1*A9BA=:(1+#C&.#(3#H=9E)AC*5,AC#E'536*2?)H14?>9'B=7(3H/B:+A:8%1-+#(E%&$$&14"76D?>7(&20H5%*&CF8!G5B+A4F$7(:"'?0$?G+$)B-?2<0<F=D!38BH,%=8&5@+ AS:i:-13    XN:i:0  XM:i:2  XO:i:1  XG:i:2  NM:i:4  MD:Z:72^TT55C15A47  YT:Z:UUr7  16  gi|9626243|ref|NC_001416.1| 4692    42  143M    *   0   0   TCAGCCGGACGCGGGCGCTGCAGCCGTACTCGGGGATGACCGGTTACAACGGCATTATCGCCCGTCTGCAACAGGCTGCCAGCGATCCGATGGTGGACAGCATTCTGCTCGATATGGACANGCCCGGCGGGATGGTGGCGGGG -"/@*7A0)>2,AAH@&"%B)*5*23B/,)90.B@%=FE,E063C9?,:26$-0:,.,1849'4.;F>FA;76+5&$<C":$!A*,<B,<)@<'85D%C*:)30@85;?.B$05=@95DCDH<53!8G:F:B7/A.E':434> AS:i:-6 XN:i:0  XM:i:2  XO:i:0  XG:i:0  NM:i:2  MD:Z:98G21C22   YT:Z:UU

The first few lines (beginning with @) are SAM header lines, and the rest of the lines are SAM alignments, one line per read or mate. See the Bowtie 2 manual section on SAM output and the SAM specification for details about how to interpret the SAM file format.

Paired-end example

To align paired-end reads included with Bowtie 2, stay in the same directory and run:

$BT2_HOME/bowtie2 -x lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam

This aligns a set of paired-end reads to the reference genome, with results written to the file eg2.sam.

Local alignment example

To use local alignment to align some longer reads included with Bowtie 2, stay in the same directory and run:

$BT2_HOME/bowtie2 --local -x lambda_virus -U $BT2_HOME/example/reads/longreads.fq -S eg3.sam

This aligns the long reads to the reference genome using local alignment, with results written to the file eg3.sam.

Using SAMtools/BCFtools downstream

SAMtools is a collection of tools for manipulating and analyzing SAM and BAM alignment files. BCFtools is a collection of tools for calling variants and manipulating VCF and BCF files, and it is typically distributed with SAMtools. Using these tools together allows you to get from alignments in SAM format to variant calls in VCF format. This example assumes that samtools andbcftools are installed and that the directories containing these binaries are in your PATH environment variable.

Run the paired-end example:

$BT2_HOME/bowtie2 -x $BT2_HOME/example/index/lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam

Use samtools view to convert the SAM file into a BAM file. BAM is a the binary format corresponding to the SAM text format. Run:

samtools view -bS eg2.sam > eg2.bam

Use samtools sort to convert the BAM file to a sorted BAM file.

samtools sort eg2.bam eg2.sorted

We now have a sorted BAM file called eg2.sorted.bam. Sorted BAM is a useful format because the alignments are (a) compressed, which is convenient for long-term storage, and (b) sorted, which is conveneint for variant discovery. To generate variant calls in VCF format, run:

samtools mpileup -uf $BT2_HOME/example/reference/lambda_virus.fa eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf

Then to view the variants, run:

bcftools view eg2.raw.bcf

】】】】