使用ChIPseeker对找到的peaks文件进行注释

from Jimmy

读入peaks

peak <- readPeakFile("RNAPII_8WG16_peaks.narrowPeak") ##一定要是narrowPeak文件

## loading packages
library(ChIPseeker)
library(clusterProfiler) 

there are 26029 peaks for this data

peaks性质

ChIP Peaks over Chromosomes

首先查看这些peaks在各个染色体上的分布，全局浏览

#peaks在所有染色体上的分布
covplot(peak,weightCol="V5")
#只看peaks在chr1，chr2上的分布
covplot(peak, chr = c("chr1", "chr2"))

image.png

peaks在所有基因的启动子附近的分布情况

library(TxDb.Mmusculus.UCSC.mm10.knownGene)
txdb <- TxDb.Mmusculus.UCSC.mm10.knownGene 
promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)
#定义tss上下游的距离
tagMatrix <- getTagMatrix(peak, windows=promoter)
tagHeatmap(tagMatrix, xlim=c(-3000, 3000), color="red")

image.png

热图每一行代表一个基因，展示的是所有基因TSS两侧的分布，除了热图外，还可以对所有基因取均值，用折线图来展示TSS两侧分布情况，用法如下

The Average Profile of ChIP peaks binding to TSS region

查看peaks在所有基因的启动子附近的分布情况，信号强度曲线图

plotAvgProf(tagMatrix, xlim=c(-3000, 3000), 
           xlab="Genomic Region (5' -> 3')", ylab = "Read Count Frequency")

image.png

peaks的注释

peakAnno <- annotatePeak(peak, tssRegion=c(-3000,3000),
                         TxDb=txdb, annoDb="org.Mm.eg.db")
peakAnno_df <- as.data.frame(peakAnno)
sampleName=strsplit(bedPeaksFile, '\\.')[1][1]
print(sampleName)

[1]"RNAPII_peaks"

write.csv(peakAnno_df, paste0(sampleName,'_peakAnno_df.csv'))
DT::datatable(peakAnno_df,
              extensions='FixedColumns',
              options =list(#dom='t', 
              scrollX =TRUE, fixedColumns = TRUE))

image.png

可以对peaks的性质做一些可视化，如下：

#png ('Pie-summarize the distribution of peaks over different type of features.png')
plotAnnoPie(peakAnno)

Screenshot 2020-02-21 at 3.40.31 PM.png

#png('Bar-summarize the distribution of peaks over different type of features.png')
plotAnnoBar(peakAnno)

Screenshot 2020-02-21 at 3.40.56 PM.png

#png('vennpie-summarize the distribution of peaks over different type of features.png')
#vennpie(peakAnno)

还可以查看peaks的长度分布，只统计长度在1000bp以下的peaks

peaksLength=abs(peakAnno_df$end-peakAnno_df$start)
peaksLength=peaksLength[peaksLength<1000]
hist(peaksLength, breaks =50, col = "lightblue", xlim=c(0,1000), xlab = "peak length", main="Histogram of peak length")

Screenshot 2020-02-21 at 3.41.23 PM.png

peaks相关基因的注释

peak的注释用annotatePeak，TSS (transcription start site) region 可以自己设定，默认是（-3000，3000），TxDb 是指某个物种的基因组，
可以把peaks先分类再注释，也可以直接拿所有peaks相关基因去富集分析，如果要分类，可以根据：
Promoter
5'UTR
3'UTR
Exon
Intron
Downstream
Intergenic
但是如果peaks本来就不多，那么分类之后基因太少，注释可能并没有意义。

genes=unique(peakAnno_df$ENSEMBL) #去除重复基因