R-loop数据分析之R-ChIP(样本间BAM比较和可视化)

样本间相关性评估

上一步得到各个样本的BAM文件之后，就可以在全基因组范围上看看这几个样本之间是否有差异。也就是先将基因组分成N个区间，然后用统计每个区间上比对上的read数。

脚本scripts/07.genome_bin_read_coverage.sh如下

#!/bin/bash

set -e
set -u
set -o pipefail

samples=${1?missing sample file}
chromsize=${2:-index/hg19.chrom.sizes}
size=${3:-3000}
ALIGN_DIR="analysis/2-read-align"
COV_DIR="analysis/3-genome-coverage"

mkdir -p ${COV_DIR}

exec 0< $samples

while read sample
do
    bedtools makewindows -g $chromsize -w $size | \
      bedtools intersect -b ${ALIGN_DIR}/${sample}.flt.bam -a - -c -bed > ${COV_DIR}/${sample}.ReadsCoverage
done

准备一个输入文件存放待处理样本的前缀，然后运行脚本bash scripts/07.genome_bin_read_coverage.sh samples_rep.txt

HKE293-D210N-Input-Rep1
HKE293-D210N-Input-Rep2
HKE293-D210N-Input-Rep3
HKE293-D210N-V5ChIP-Rep1
HKE293-D210N-V5ChIP-Rep2
HKE293-D210N-V5ChIP-Rep3

最后将得到的文件导入到R语言中进行作图，使用的是基础绘图系统的光滑散点图(smoothScatter)。

files_list <- list.files("r-chip/analysis/3-genome-coverage","ReadsCoverage")
files_path <- file.path("r-chip/analysis/3-genome-coverage",files_list)

input_rep1 <- read.table(files_path[1], sep='\t')
input_rep2 <- read.table(files_path[2], sep='\t')
input_rep3 <- read.table(files_path[3], sep='\t')
chip_rep1 <- read.table(files_path[4], sep='\t')
chip_rep2 <- read.table(files_path[5], sep='\t')
chip_rep3 <- read.table(files_path[6], sep='\t')

pw_plot <- function(x, y, 
                    xlab="x",
                    ylab="y", ...){
  log2x <- log2(x)
  log2y <- log2(y)
  smoothScatter(log2x,log2y,
                cex=1.2,
                xlim=c(0,12),ylim=c(0,12),
                xlab=xlab,
                ylab=ylab)
  text(3,10,paste("R = ",round(cor(x,y),2),sep=""))
}

par(mfrow=c(2,3))
pw_plot(chip_rep1[,4], chip_rep2[,4],
        xlab = "Rep 1 (Log2 Tag Counts)",
        ylab = "Rep 2 (Log2 Tag Counts)")

pw_plot(chip_rep1[,4], chip_rep3[,4],
        xlab = "Rep 1 (Log2 Tag Counts)",
        ylab = "Rep 2 (Log2 Tag Counts)")

pw_plot(chip_rep2[,4], chip_rep3[,4],
        xlab = "Rep 1 (Log2 Tag Counts)",
        ylab = "Rep 2 (Log2 Tag Counts)")

pw_plot(input_rep1[,4], input_rep2[,4],
        xlab = "Rep 1 (Log2 Tag Counts)",
        ylab = "Rep 2 (Log2 Tag Counts)")

pw_plot(input_rep1[,4], input_rep3[,4],
        xlab = "Rep 1 (Log2 Tag Counts)",
        ylab = "Rep 3 (Log2 Tag Counts)")

pw_plot(input_rep2[,4], input_rep3[,4],
        xlab = "Rep 2 (Log2 Tag Counts)",
        ylab = "Rep 3 (Log2 Tag Counts)")

correlationship

这种多个BAM文件之间相关性衡量，其实也可以用deepTools的plotCorrelation画出来，但是我觉得应该没有R语言画 的好看。

BigWig可视化

由于同一个样本间的BAM文件具有很强的相关性，因此可以将这些样本合并起来转换成bigwig格式，这样子在基因组浏览器（例如IGV, UCSC Browser, JBrowse）上方便展示。

如下的代码的目的就是先合并BAM，然后转换成BigWig，拆分成正链和反链进行保存

#!/bin/bash

set -e
set -u
set -o pipefail

samples=${1?please provied sample file}
threads=${2-8}
bs=${3-50}

ALIGN_DIR="analysis/2-read-align"
BW_DIR="analysis/4-normliazed-bw"

mkdir -p $BW_DIR

exec 0< $samples
cd $ALIGN_DIR

while read sample
do
    bams=$(ls ${sample}*flt.bam | tr '\n' ' ')
    if [ ! -f ../$(basename ${BW_DIR})/${sample}.tmp.bam ]; then
    echo "samtools merge -f -@ ${threads} ../$(basename ${BW_DIR})/${sample}.tmp.bam $bams  &&
          samtools index ../$(basename ${BW_DIR})/${sample}.tmp.bam" | bash
    fi
done

cd ../../
exec 0< $samples

cd ${BW_DIR}
while read sample
do
    bamCoverage -b ${sample}.tmp.bam -o ${sample}_fwd.bw -of bigwig  \
      --filterRNAstrand forward --binSize ${bs} --normalizeUsing CPM --effectiveGenomeSize 2864785220 \
      --extendReads 150 -p ${threads} 2> ../log/${sample}_fwd.log
    bamCoverage -b ${sample}.tmp.bam -o ${sample}_rvs.bw -of bigwig  \
      --filterRNAstrand reverse --binSize ${bs} --normalizeUsing CPM --effectiveGenomeSize 2864785220 \
      --extendReads 150 -p ${threads} 2> ../log/${sample}_rvs.log
    rm -f ${sample}.tmp.bam ${sample}.tmp.bam.bai
done

得到的BW文件你可以在IGV上初步看看，比如说检查下文章Figure 1(E)提到的CIRH1A基因

CIRH1A

发表文章时肯定不能用上图，我们可以用R的Gviz进行展示下

library(Gviz)

#下面将scale等track写入tracklist
tracklist<-list()
itrack <- IdeogramTrack(genome = "hg19", chromosome = 'chr16',outline=T)
tracklist[["itrack"]]<-itrack

# 读取BigWig
bw_file_path <- "C:/Users/DELL/Desktop/FigureYa/R-ChIP/"
bw_file_names <- list.files(bw_file_path, "*.bw")
bw_files <- file.path("C:/Users/DELL/Desktop/FigureYa/R-ChIP/",
                      bw_file_names)

tracklist[['D210-fwd']] <- DataTrack(range = bw_files[3],
                              genome="hg19",
                              type="histogram",
                              name='D210 + ',
                              ylim=c(0,4),
                              col.histogram="#2167a4",
                              fill.histogram="#2167a4")
tracklist[['D210-rvs']] <- DataTrack(range = bw_files[4],
                                     genome="hg19",
                                     type="histogram",
                                     name='D210 - ',
                                     ylim=c(4,0),
                                     col.histogram="#eb1700",
                                     fill.histogram="#eb1700")

tracklist[['WKDD-fwd']] <- DataTrack(range = bw_files[9],
                                     genome="hg19",
                                     type="histogram",
                                     name='WKDD + ',
                                     ylim=c(0,4),
                                     ylab=2,
                                     col.histogram="#2167a4",
                                     fill.histogram="#2167a4")
tracklist[['WKDD-rvs']] <- DataTrack(range = bw_files[10],
                                     genome="hg19",
                                     type="histogram",
                                     name=' WKDD -',
                                     ylim=c(4,0),
                                     col.histogram="#eb1700",
                                     fill.histogram="#eb1700",
                                     showAxis=TRUE)

#写入比例尺
scalebar <- GenomeAxisTrack(scale=0.25,
                            col="black",
                            fontcolor="black",
                            name="Scale",
                            labelPos="above",showTitle=F)
tracklist[["scalebar"]]<-scalebar

# 画图
plotTracks(trackList = tracklist,
           chromosome = "chr16",
           from =  69141913, to= 69205033,
           background.panel = "#f6f6f6",
           background.title = "white",
           col.title="black",col.axis="black",
           rot.title=0,cex.title=0.5,margin=10,title.width=1.75,
           cex.axis=1
           )

Gviz

后续用AI修改下坐标轴，就几乎和原图差不多了。此外可能还要调整之前脚本的bin size， 使得整体更加平滑

看图说话： 由于WKKD在D210N的基础上继续突变了几个位点，使得原本只丧失了RNASEH1的催化活性的D210N进一步丧失了结合到RNA/DNA杂合体的能力，在实验中就可以作为 负对照。上图就是其中一个有代表性的区间。