1.input normalize ## 消除背景噪音

ChIP=C57BL6_Brain_Sample_1_MeRIP-SYSY.hisat2
Input=C57BL6_Brain_Sample_1_Non-IP_Control.hisat2
operation=log2

bigwigCompare -p 50  -b1 ${ChIP}.bw  -b2 ${Input}.bw \
--operation ${operation}  --binSize 10 --pseudocount 1 \
-o ${ChIP}_${operation}_${Input}.bw --outFileFormat bigwig \
> ${ChIP}_${operation}_${Input}.log &

#——————————————————————–

2.prepare matrix

cd /home4/sjshen/project/m6a/GSE29714_Cell/1.1_hisat2/mouse/position_sorted_bam/deeptools_profile/result

bw_dir1=/home4/sjshen/project/m6a/GSE29714_Cell/1.1_hisat2/mouse/position_sorted_bam/deeptools_bw/assemble_bw
region_dir1=/home4/sjshen/project/m6a/GSE29714_Cell/1.1_hisat2/mouse/position_sorted_bam/deeptools_profile/bed_home

bw_files="
$bw_dir1/C57BL6_Brain_Sample_1_MeRIP-SYSY.hisat2_log2ratio_C57BL6_Brain_Sample_1_Non-IP_Control.hisat2.bw 
$bw_dir1/C57BL6_Brain_Sample_2_MeRIP-NEB.hisat2_log2ratio_C57BL6_Brain_Sample_2_Non-IP_Control.hisat2.bw 
$bw_dir1/C57BL6_Brain_Sample_2_MeRIP-SYSY.hisat2_log2ratio_C57BL6_Brain_Sample_2_Non-IP_Control.hisat2.bw 
"
region_files="
$region_dir1/mm10.RefSeq.cds.bed
"

try=MeIRP_profile_on_cds_mm10_input_norm

nohup computeMatrix scale-regions -p 100  -b 1000 -a 1000 --regionBodyLength 3000 -R $region_files \
-S $bw_files --startLabel cdsStart  --endLabel cdsEnd --binSize 50 \
--samplesLabel  SYSY-MeRIP-rep1 NEB-MeRIP-rep2 SYSY-MeRIP-rep2 \
-o ./${try}_computeMatrix.gz --outFileNameMatrix ./${try}_computeMatrix.matrix.tab \
--outFileSortedRegions ./${try}_computeMatrix.SortedRegions.bed > ./${try}_computeMatrix.log &

#——————————————————————–

3.display graph

try=MeIRP_profile_on_cds_mm10_input_norm

plotProfile --matrixFile ${try}_computeMatrix.gz --outFileName ${try}_profile_1.pdf --outFileNameData ${try}_profile_1.tab --dpi 720 --perGroup --numPlotsPerRow 2 --plotHeight 12 --plotWidth 16 &
plotProfile --matrixFile ${try}_computeMatrix.gz --outFileName ${try}_profile_2.pdf --outFileNameData ${try}_profile_2.tab --dpi 720            --numPlotsPerRow 2 --plotHeight 12 --plotWidth 16 &

Tips：主要查看基因CDS区域上带有m6a的reads的分布

也可以展示每个基因的情况

plotHeatmap --matrixFile ./${try}_computeMatrix.gz --outFileName ./${try}_peak.pdf --colorList "white,blue" --whatToShow "plot, heatmap and colorbar" --dpi 720 --missingDataColor white \
--refPointLabel "center" --regionsLabel " "  --xAxisLabel " " --sortRegions descend --sortUsing mean --heatmapHeight 30 --heatmapWidth 4 &

Dependencies

• Softwares
  • deeptools