一个超简单的转录组项目全过程--iMac+RNA-Seq（四）f

好久没更新啦，因为最近没有在跑项目哦，所以就搁置了，最近要做一两张图，所以把前面写的教程先发布一下~

前期文章

一个超简单的转录组项目全过程--iMac+RNA-Seq（一）
一个超简单的转录组项目全过程--iMac+RNA-Seq（二）QC
一个超简单的转录组项目全过程--iMac+RNA-Seq（三）Alignment 比对

4 featureCounts

比对结束后需要进行计数咯！

老规矩，先看说明书。

featureCounts -h ## 好像发现了什么不得了的
$featureCounts -h
featureCounts: invalid option -h

Version 1.6.2

Usage: featureCounts [options] -a <annotation_file> -o <output_file> input_file1 [input_file2] ...

-T number of threads

-p If specified, fragments (or templates) will be counted instead of reads. This option is only applicable for paired-end reads.

-t <string> Specify feature type in GTF annotation. 'exon' by default. Features used for read counting will be extracted from annotation using the provided value.

-g <string> Specify attribute type in GTF annotation. 'gene_id' by default. Meta-features used for read counting will be extracted from annotation using the provided value.

-a <string> Name of an annotation file. GTF/GFF format by default. See -F option for more format information. Inbuilt annotations (SAF format) is available in 'annotation' directory of the package. Gzipped file is also accepted.

-o <string> Name of the output file including read counts. A separate file including summary statistics of counting results is also included in the output ('<string>.summary')

用法一

## 教程代码，使用for循环
mkdir $wkd/align   #这样会生成单个的，每个都有自己的名字
cd $wkd/align 

source activate rna 
for fn in {512..520} 
do 
featureCounts -T 4 -p -t exon -g gene_id \
-a /Users/bioinformatic/reference/hg38/hg38.gtf \
-o counts.SRR1039$fn.txt /home/rna/clean/SRR1039$fn.hisat.bam 
done 
source deactivate 

用法二：酷酷的

vim count.sh ## 创建一个脚本叫做count

#!/bin/bash
set -e
set -u
set -o pipefail

# set PATH 设置路径
HUMAN=/Users/bioinformatic/reference/hg38
OUTDIR=/Users/Desktop/project/clean/align

# source activate rna
cd $OUTDIR  # 打开目标文件夹

pwd  ## 显示当前路径

ls *bam | cut -d"_" -f 1| sort -u |\
while read id 
do   # 用echo语句来提示脚本运行进度
     echo "processing  ${id}_combined.hisat.bam" 
     if [ ! -f ${id}.hisat.done ] 
     then
     featureCounts -T 4 -p \
         -t exon -g gene_id -a $HUMAN/hg38.gtf \
         -o counts.${id}.txt \
         $OUTDIR/${id}_combined.hisat.bam && touch ${id}.hisat.done
      fi

done

# source deactivate 

Note：脚本很长的时候，记得使用“\”符号加回车，让行文变得整洁易读。

运行脚本

nohup bash count.sh &

查看结果

cat nohup.out就可以看到featureCounts的运行日志，请注意以下两个数据

26834745 (92.89%) aligned concordantly exactly 1 time
97.89% overall alignment rate

小结：本章所需知识背景和技能

（1）基因表达量的计算到底在算什么？

（2）TPM, RPKM, FPKM这几个计量方式的区别

（3）学会查看帮助文档，了解软件用法

（4）nohup是什么，nohup和&的区别等等

用法二的知识点

在酷酷的用法二里面有几个很酷的地方：

#!/bin/bash
set -e
set -u
set -o pipefail

# touch的用法

# if then fi 语句的使用

shell脚本和正则表达式：必学