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
I wouldn't expect the peak heights to be identical, some amount of biological variation is normal. The goal of the normalization should instead be to set the background level to roughly similar values between samples.

You do not need to normalize your BAM files, CSAW or DiffBind will take care of that step for you.

If you want to downsample you can either use `samtools view -s` if starting from BAM files or `seqtk` if starting from fastq files. This is generally not needed.