## step5 : alignment to hg19/ using bowtie2 to do alignment
## ~/biosoft/bowtie/bowtie2-2.2.9/bowtie2-build ~/biosoft/bowtie/hg19_index /hg19.fa ~/biosoft/bowtie/hg19_index/hg19
## cat >run_bowtie2.sh
ls *.fastq | while read id ;
do
echo $id
#~/biosoft/bowtie/bowtie2-2.2.9/bowtie2 -p 8 -x ~/biosoft/bowtie/hg19_index/hg19 -U $id -S ${id%%.*}.sam 2>${id%%.*}.align.log;
#samtools view -bhS -q 30 ${id%%.*}.sam > ${id%%.*}.bam ## -F 1548 https://broadinstitute.github.io/picard/explain-flags.html
# -F 0x4 remove the reads that didn't match
samtools sort ${id%%.*}.bam ${id%%.*}.sort ## prefix for the output
# samtools view -bhS a.sam | samtools sort -o - ./ > a.bam
samtools index ${id%%.*}.sorted.bam
done

这个索引~/biosoft/bowtie/hg19_index/hg19需要自己提取建立好，见前文

初步比对的sam文件到底该如何过滤，我查了很多文章都没有给出个子丑寅卯，各执一词，我也没办法给大家一个标准，反正我测试了好几种，看起来call peaks的差异不大，就是得不到文章给出的那些结果！！

一般来说，初步比对的sam文件只能选取unique mapping的结果，所以我用了#samtools view -bhS -q 30，但是结果并没什么改变，有人说是peak caller这些工具本身就会做这件事，所以取决于你下游分析所选择的工具。

给大家看比对的日志吧：

SRR1042593.fastq
16902907 reads; of these:
16902907 (100.00%) were unpaired; of these:
667998 (3.95%) aligned 0 times
12467095 (73.76%) aligned exactly 1 time
3767814 (22.29%) aligned >1 times
96.05% overall alignment rate
[samopen] SAM header is present: 93 sequences.
SRR1042594.fastq
60609833 reads; of these:
60609833 (100.00%) were unpaired; of these:
9165487 (15.12%) aligned 0 times
39360173 (64.94%) aligned exactly 1 time
12084173 (19.94%) aligned >1 times
84.88% overall alignment rate
[samopen] SAM header is present: 93 sequences.
SRR1042595.fastq
14603295 reads; of these:
14603295 (100.00%) were unpaired; of these:
918028 (6.29%) aligned 0 times
10403045 (71.24%) aligned exactly 1 time
3282222 (22.48%) aligned >1 times
93.71% overall alignment rate
[samopen] SAM header is present: 93 sequences.
SRR1042596.fastq
65911151 reads; of these:
65911151 (100.00%) were unpaired; of these:
10561790 (16.02%) aligned 0 times
42271498 (64.13%) aligned exactly 1 time
13077863 (19.84%) aligned >1 times
83.98% overall alignment rate
[samopen] SAM header is present: 93 sequences.
SRR1042597.fastq
22210858 reads; of these:
22210858 (100.00%) were unpaired; of these:
1779568 (8.01%) aligned 0 times
15815218 (71.20%) aligned exactly 1 time
4616072 (20.78%) aligned >1 times
91.99% overall alignment rate
[samopen] SAM header is present: 93 sequences.
SRR1042598.fastq
58068816 reads; of these:
58068816 (100.00%) were unpaired; of these:
8433671 (14.52%) aligned 0 times
37527468 (64.63%) aligned exactly 1 time
12107677 (20.85%) aligned >1 times
85.48% overall alignment rate
[samopen] SAM header is present: 93 sequences.
SRR1042599.fastq
24019489 reads; of these:
24019489 (100.00%) were unpaired; of these:
1411095 (5.87%) aligned 0 times
17528479 (72.98%) aligned exactly 1 time
5079915 (21.15%) aligned >1 times
94.13% overall alignment rate
[samopen] SAM header is present: 93 sequences.
SRR1042600.fastq
76361026 reads; of these:
76361026 (100.00%) were unpaired; of these:
8442054 (11.06%) aligned 0 times
50918615 (66.68%) aligned exactly 1 time
17000357 (22.26%) aligned >1 times
88.94% overall alignment rate
[samopen] SAM header is present: 93 sequences.

可以看到比对非常成功！！！我这里就不用表格的形式来展现了，毕竟我又不是给客户写报告，大家就将就着看吧。

Overview:

MOSAIK is a stable, sensitive and open-source program for mapping second and 
third-generation sequencing reads to a reference genome. Uniquely among current 
mapping tools, MOSAIK can align reads generated by all the major sequencing 
technologies, including Illumina, Applied Biosystems SOLiD, Roche 454, 
Ion Torrent and Pacific BioSciences SMRT.

一，软件安装

二，输入数据准备

三，运行命令

这两个步骤是构建hash索引，对这个60M的压缩包病毒基因组集合，时间是

MosaikBuild CPU time: 15.660 s, wall time: 18.146 s

MosaikJump CPU time: 329.031 s, wall time: 331.672 s

还可以接受，但是输出的index文件就有点难以接受了！！！！

333M Mar 11 19:55 gibVirus.fa.bin

60M Aug 13  2013 gibVirus.fa.gz

5.0G Mar 11 20:04 gibVirus.JumpDb_keys.jmp

1 Mar 11 19:59 gibVirus.JumpDb_meta.jmp

1.3G Mar 11 20:04 gibVirus.JumpDb_positions.jmp

如果是对人的hg19基因组来说，消耗的时间如下：

MosaikBuild CPU time: 183.642 s, wall time: 184.658 s

MosaikJump CPU time: 3985.608 s, wall time: 3995.323 s

一个多小时，还行！

对参考基因组建好了索引，还需要对测序数据构建索引！

数据双端测序，每个1.6G左右数据，构建索引耗时如下：

接下来就比对！

比对的结果就是那个L526401A.bin.aligned，但是还需要用MosaikText转换成sam格式方便阅读！

其实它github里面有测试数据，你跑一遍就懂了！

四，数据结果解读