網(wǎng)站：https://sourceforge.net/projects/apatrap/
說明書：https://sourceforge.net/p/apatrap/wiki/User%20Manual/
1.fatsq的QC
2.fastq做trim
3.比對：需要用轉(zhuǎn)錄本：
hisat2 網(wǎng)站上下載的GRCm38.fasta.tran.index
這是ensembl的數(shù)據(jù)躺翻，比對后給他加"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轉(zhuǎn)換成bam：
sort不需要加n 正常sort即可弧可！
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.轉(zhuǎn)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
6.提取UTR:
參數(shù)：
'/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
#?genesymbol文件如下：
下載ref：轉(zhuǎn)錄本的bed文件下載 - 簡書
這里有個致命出錯：就是這個genesymbol竟然是以空格分隔的罗丰！用awk改為\t分隔疟暖。
# 此處mm10的refbed時ensembl：
下載ref：轉(zhuǎn)錄本的bed文件下載 - 簡書
7.生成APA：
參數(shù)：
'/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
8.R進(jìn)行差異APA分析：
用法：deAPA(input_file, output_file, group1, group2, least_qualified_num_in_group1, least_qualified_num_in_group2, coverage_cutoff)
library('deAPA')
deAPA('APA.txt', 'APA.result.txt', 2, 2, 1, 1, 20)
