如何获得FPKM/RPKM计算需要的基因长度（考虑exon之间的overlap）

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这里我们跟Cufflinks的原理一致，使用总的外显子长度，并且去除过多的重叠的外显子的部分。使用R语言,输入为基因的GTF文件

包的安装

依赖data.table, IRanges,rtracklayer

install.packages("data.table")
if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("rtracklayer")
BiocManager::install("IRanges")

代码

library(data.table)
library("IRanges")
require("rtracklayer")

hg19 <- readGFF("hg19.gencodev27.gtf")
anno <- setDT(hg19)
anno <- anno[type=="exon",]
setnames(anno,c("seqid","start","end","gene_name","exon_number"),c("Chr","ExonStart","ExonEnd","Gene","Exon_number"))
#mkdir bin and mean by bin
Exon_region <- unique(anno[,.(Chr,ExonStart,ExonEnd,Exon_number,Gene)])
Exon_region <- Exon_region[,{x <- IRanges(ExonStart,ExonEnd);y <- reduce(x); list(ExonStart=y@start,ExonEnd=y@start+y@width-1)},by=.(Gene,Chr)]
Exon_region[,Exon_num:=1:.N,by=Gene]
Exon_region <- Exon_region[,.(Chr,ExonStart,ExonEnd,Exon_num,Gene)]
Exon_len <- Exon_region[,.(ExonLen = ExonEnd - ExonStart + 1),by=.(Exon_num,Gene)]
gene_len <- Exon_len[,.(Length = sum(ExonLen)),by=Gene]


# write out
fwrite(Exon_region,file="All_hg19gene_exon.bed", sep = "	", col.names = T)
fwrite(gene_len, file = "All_hg19gene_len.txt", sep = "	", col.names = T)
~

结果文件

1. 基因长度文件 链接：https://pan.baidu.com/s/1NtfM_ESyNyaT-kVaKu0MyQ
   提取码：gy88
   复制这段内容后打开百度网盘手机App，操作更方便哦

2. 合并后的外显子区域文件
   链接：https://pan.baidu.com/s/1-IpuC_2N88Jx9m2g5fCqmA
   提取码：cevo
   复制这段内容后打开百度网盘手机App，操作更方便哦

参考资料

https://www.cureffi.org/2013/09/12/counts-vs-fpkms-in-rna-seq/