轉錄組入門（5）：序列比對

比對軟件很多，首先大家去收集一下，因為我們是帶大家入門，請統一用hisat2，并且搞懂它的用法。
直接去hisat2的主頁下載index文件即可，然后把fastq格式的reads比對上去得到sam文件。
接著用samtools把它轉為bam文件，并且排序(注意N和P兩種排序區別)索引好，載入IGV，再截圖幾個基因看看！
順便對bam文件進行簡單QC，參考直播我的基因組系列。

HISAT2安裝：

linux版Hisat2下載，解壓，可以使用了：
$ wget ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/downloads/hisat2-2.1.0-Linux_x86_64.zip
解壓（-d 解壓到指定文件）：
$ unzip -d /work/LXJ/software/ hisat2-2.1.0-Linux_x86_64.zip
檢查是否可以運行：
$ ./hisat2
(ERR): hisat2-align exited with value 1：可以忽略

環境路徑設置:
$ sudo vi /etc/environment
添加：/work/LXJ/software/hisat2-2.1.0
$ source /etc/environment

HISAT2使用

基因組索引

自行建立基因組索引：
Command Line ： hisat2-build [options]* <reference_in> <ht2_base>
Usage : hisat2-build –p 8 genome.fa genome
如果想分析關于snp、exon、剪切位點新的信息,HISAT2建立基因組索引時，需要加入注釋過的snp、exon、剪切位點后，再信息建立基因組索引；(hisat2包中有程序幫你解決)
下載基因組索引：
從HISAT2的官網中下載現成的基因組索引，這樣子比較省事，也可以防止出錯：

這是老鼠的基因組索引，根據需要下載合適的版本：
$ wget ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/mm10.tar.gz tar zxvf mm10.tar.gz

HISAT2比對RNA-Seq到基因組：
hisat2 [options]* -x <hisat2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]
<ht2-idx> Index filename prefix (minus trailing .X.ht2).
<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).
<SRA accession number> Comma-separated list of SRA accession numbers, e.g. --sra-acc SRR353653,SRR353654.
<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'.

HISAT2比對：

for i in {59..62};
do
echo $i
hisat2 -t -p 8 -x /work/LXJ/Genome/M.musculus/mm10.hisat2.index/genome -1 SRR35899${i}.sra_1.fastq.gz -2 SRR35899${i}.sra_2.fastq.gz -S /mnt/hgfs/Labubuntu_data/GSE81916.RNAseq/hisat2.mm10/SRR35899${i}.sam;
done

59
Time loading forward index: 00:00:25
Time loading reference: 00:00:04
Multiseed full-index search: 00:15:41
30468155 reads; of these:
  30468155 (100.00%) were paired; of these:
    2722598 (8.94%) aligned concordantly 0 times
    24300848 (79.76%) aligned concordantly exactly 1 time
    3444709 (11.31%) aligned concordantly >1 times
    ----
    2722598 pairs aligned concordantly 0 times; of these:
      156872 (5.76%) aligned discordantly 1 time
    ----
    2565726 pairs aligned 0 times concordantly or discordantly; of these:
      5131452 mates make up the pairs; of these:
        3276583 (63.85%) aligned 0 times
        1334447 (26.01%) aligned exactly 1 time
        520422 (10.14%) aligned >1 times
94.62% overall alignment rate
Time searching: 00:15:45
Overall time: 00:16:11
60
Time loading forward index: 00:00:29
Time loading reference: 00:00:04
Multiseed full-index search: 00:29:01
52972617 reads; of these:
  52972617 (100.00%) were paired; of these:
    4438954 (8.38%) aligned concordantly 0 times
    42836426 (80.87%) aligned concordantly exactly 1 time
    5697237 (10.76%) aligned concordantly >1 times
    ----
    4438954 pairs aligned concordantly 0 times; of these:
      268939 (6.06%) aligned discordantly 1 time
    ----
    4170015 pairs aligned 0 times concordantly or discordantly; of these:
      8340030 mates make up the pairs; of these:
        5335211 (63.97%) aligned 0 times
        2173091 (26.06%) aligned exactly 1 time
        831728 (9.97%) aligned >1 times
94.96% overall alignment rate
Time searching: 00:29:05
Overall time: 00:29:34
61
Time loading forward index: 00:00:31
Time loading reference: 00:00:05
Multiseed full-index search: 00:21:39
36763726 reads; of these:
  36763726 (100.00%) were paired; of these:
    3102153 (8.44%) aligned concordantly 0 times
    29382458 (79.92%) aligned concordantly exactly 1 time
    4279115 (11.64%) aligned concordantly >1 times
    ----
    3102153 pairs aligned concordantly 0 times; of these:
      173349 (5.59%) aligned discordantly 1 time
    ----
    2928804 pairs aligned 0 times concordantly or discordantly; of these:
      5857608 mates make up the pairs; of these:
        3596954 (61.41%) aligned 0 times
        1595531 (27.24%) aligned exactly 1 time
        665123 (11.35%) aligned >1 times
95.11% overall alignment rate
Time searching: 00:21:44
Overall time: 00:22:15
62
Time loading forward index: 00:00:28
Time loading reference: 00:00:05
Multiseed full-index search: 00:22:33
43802631 reads; of these:
  43802631 (100.00%) were paired; of these:
    3816434 (8.71%) aligned concordantly 0 times
    35462440 (80.96%) aligned concordantly exactly 1 time
    4523757 (10.33%) aligned concordantly >1 times
    ----
    3816434 pairs aligned concordantly 0 times; of these:
      209180 (5.48%) aligned discordantly 1 time
    ----
    3607254 pairs aligned 0 times concordantly or discordantly; of these:
      7214508 mates make up the pairs; of these:
        4769954 (66.12%) aligned 0 times
        1806461 (25.04%) aligned exactly 1 time
        638093 (8.84%) aligned >1 times
94.56% overall alignment rate
Time searching: 00:22:38
Overall time: 00:23:06

Samtools

samtools view:

Sam文件轉換為bam文件：

for i in {59..62};
do
echo $i
samtools view -S SRR35899${i}.sam -b > SRR35899${i}.bam;
done

samtools sort:

sort對bam文件排序，而不是sam文件；對比對結果按reads名稱排序（默認根據染色體上對應位置排序）;此處依據reads名字排序是為了滿足后面HTseq的計算，如果此處使用默認的chr position會增大HTseq生成count文件時的工作量。

for i in {59..62};
do
echo $i
samtools sort -n SRR35899${i}.bam -@ 8 SRR35899${i}_n.sorted;
done

默認按照染色體位置進行排序，而-n參數則是根據read名進行排序; -t，首先根據tag TAG排序，然后根據染色體位置或reads名字排序。

IGV查看

比對結果質控：
常用工具有
Picard https://broadinstitute.github.io/picard/
RSeQC http://rseqc.sourceforge.net/
Qualimap http://qualimap.bioinfo.cipf.es/
此處使用RseQC，RseQC下屬各式各樣的工具，并且RseQC官網中有測試數據和運行實例
RseQC
安裝：pip install RseQC
可使用程序：

參考：
轉錄組入門（5）： 序列比對

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