Bam to bw and metaplot

1. index bam files

for file in ~/chipseq/results/bowtie2/*aln.bam
do
samtools index $file
done

2. Bam to be files

a. Direct conversion (no input)

bamCoverage -b bowtie2/H1hesc_Nanog_Rep2_aln.bam \
-o visualization/bigWig/H1hesc_Nanog_Rep2.bw \
--binSize 20 \
--normalizeUsing BPM \
--smoothLength 60 \
--extendReads 150 \
--centerReads \
-p 6 2> ../logs/Nanog_rep2_bamCoverage.log

b. Normalization with input

bamCompare -b1 bowtie2/H1hesc_Pou5f1_Rep1_aln.bam \
-b2 bowtie2/H1hesc_Input_Rep1_chr12_aln.bam \
-o visualization/bigWig/H1hesc_Pou5f1_Rep1_bgNorm.bw \
--binSize 20 \
--normalizeUsing BPM \
--smoothLength 60 \
--extendReads 150 \
--centerReads \
-p 6 2> ../logs/Pou5f1_rep1_bamCompare.log

3. Profile plots and heatmaps

a: Creat intermediate .gz files

computeMatrix reference-point --referencePoint TSS \
-b 1000 -a 1000 \
-R ~/chipseq/results/visualization/chr12_genes.bed \
-S /n/groups/hbctraining/chip-seq/full-dataset/bigWig/Encode_Nanog*.bw \
--skipZeros \
-o ~/chipseq/results/visualization/matrixNanog_TSS_chr12.gz \
-p 6 \
--outFileSortedRegions ~/chipseq/results/visualization/regions_TSS_chr12.bed

b. Plot the metablots---Reference point

plotProfile -m visualization/matrixNanog_TSS_chr12.gz \
-out visualization/figures/TSS_Nanog_profile.png \
--perGroup \
--colors green purple \
--plotTitle "" --samplesLabel "Rep1" "Rep2" \
--refPointLabel "TSS" \
-T "Nanog read density" \
-z ""

image.png

Alternatively, we could use a heatmap to evaluate the same matrix of information:

 plotHeatmap -m visualization/matrixNanog_TSS_chr12.gz \
-out visualization/figures/TSS_Nanog_heatmap.png \
--colorMap RdBu \
--whatToShow 'heatmap and colorbar' \
--zMin -4 --zMax 4  

image.png

If we wanted both images in one single plot, we can do that with plotHeatmap and just removing the --whatToShow parameter.

plotHeatmap -m visualization/matrixPou5f1_TSS_chr12.gz \
-out visualization/figures/TSS_Pou5f1_profile-heatmap.png \
--colorMap RdBu \
--zMin -2 --zMax 2  

image.png

c. Plot the metablots---Scaled region

 computeMatrix scale-regions \
-R ~/chipseq/results/visualization/Nanog_enriched.bed \
-S /n/groups/hbctraining/chip-seq/full-dataset/bigWig/Encode_Pou5f1*.bw /n/groups/hbctraining/chip-seq/full-dataset/bigWig/Encode_Nanog*.bw \
--skipZeros \
-p 6 \
--regionBodyLength 2000 \
-a 500 -b 500 \
-o ~/chipseq/results/visualization/matrixAll_Nanog_binding_sites.gz


 plotProfile -m visualization/matrixAll_Nanog_binding_sites.gz \
-out visualization/figures/Allsamples_NanogSites_profile.png \
--perGroup  --plotTitle "" \
--samplesLabel "Pou5f1-Rep1" "Pou5f1-Rep2" "Nanog-Rep1" "Nanog-Rep2" \
-T "Nanog only binding sites"  -z "" \
--startLabel "" \
--endLabel "" \
--colors red red darkblue darkblue

Note: Here enriched region were selected. We can also use the bed files of all genes to get a genome wide metaplot

image.png

Ref to：https://hbctraining.github.io/Intro-to-ChIPseq/lessons/10_data_visualization.html