APAtrap的使用

网站：https://sourceforge.net/projects/apatrap/

1.fatsq的QC

2.fastq做trim

3.比对：需要用转录本：

hisat2 网站上下载的GRCm38.fasta.tran.index

这是ensembl的数据，比对后给他加"chr"

hisat2 -x $index -p 10 -1 T1-R1.fastq.gz -2 T1-R2.fastq.gz -S T1.sam --add-chrname

hisat2 -x $index -p 10 -1 T2-R1.fastq.gz -2 T2-R2.fastq.gz -S T2.sam --add-chrname

hisat2 -x $index -p 10 -1 WT1-R1.fastq.gz -2 WT1-R2.fastq.gz -S WT1.sam --add-chrname

hisat2 -x $index -p 10 -1 WT2-R1.fastq.gz -2 WT2-R2.fastq.gz -S WT2.sam --add-chrname

4.samtools转换成bam：

samtools sort --threads 20 -m 3G -o T-1.bam T-1.sam

samtools sort --threads 20 -m 3G -o T-2.bam T-2.sam

samtools sort --threads 20 -m 3G -o WT-1.bam WT-1.sam

samtools sort --threads 20 -m 3G -o WT-2.bam WT-2.sam

5.转成bedgraph：

genomeCoverageBed -bg -ibam T-1.bam -split > T-1.bedgraph

genomeCoverageBed -bg -ibam T-2.bam -split > T-1.bedgraph

genomeCoverageBed -bg -ibam WT-1.bam -split > WT-1.bedgraph

genomeCoverageBed -bg -ibam WT-2.bam -split > WT-2.bedgraph

head查看bedgraph

6.提取UTR:

参数：

/home/pc/biosoft/APAtrap/identifyDistal3UTR

'/home/pc/biosoft/APAtrap/identifyDistal3UTR' -s $genesymbol -i T-1.bedgraph T-2.bedgraph WT-1.bedgraph WT-2.bedgraph -m mm10.refgene.bed -o UTR.bed

head查看下UTR.bed

# 此处mm10的refbed时ensembl

下载ref：转录本的bed文件下载 - 简书

head查看下mm10.refgene.bed head查看genesymbol

7.生成APA：

参数：

/home/pc/biosoft/APAtrap/predictAPA

'/home/pc/biosoft/APAtrap/predictAPA' -i T-1.bedgraph T-2.bedgraph WT-1.bedgraph WT-2.bedgraph -g 2 -n 2 2 -u UTR.bed -o APA.txt

head查看下APA.txt

8.R进行差异APA分析：

library('deAPA')

deAPA('APA.txt', 'APA.result.txt', 1, 2, 1, 1, 20)

head查看下APA.result.txt