I have paired-end 4 ATAC-seq data (2 replicates for 2 samples). I have done aligning using Bowtie2. I did filter MT reads and duplicates using Picard, then performed peak calling on Bam file using MACS2. Also I did differential peak analysis using deeptools and filter them by FDR<0.05 and abs(2foldchange)>2.
After these, I generated density peak heatmaps using deeptools. However, on the figure top on the heatmaps height of peaks are not the same for 4 files although I normalized bam files while converting to bigwig using bamCoverage.
My questions are: Should the height of those peaks be the same or slight change is acceptable? If not how can I normalize the data? Should I normalize bam files then do the peak calling again if so which tool you suggest? or diffbind normalization okay? Also, I am really confused about the coverage file normalization and peak normalization. Lastly, as written in this post Normalization and differential analysis in ATAC-seq data how can I downsample each sample?
If you could explain these I will appreciate it.