Hi all,

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 https://www.biostars.org/p/308976/ how can I downsample each sample?

If you could explain these I will appreciate it. 





![peak signal heatmap][1]


  [1]: https://www.dropbox.com/home?preview=image.png
In some cases normalizing only for sequencing depth might be enough. Often it is not due to differences in library composition and different signal-to-noise ratios. I prefer to scale my bigwig files (or whatever counts you want to normalize) with the normalization factors from `edgeR`. Code examples and some details in the linked thread: https://www.biostars.org/p/413626/#414440