Is the MACS pileup output normalized

Question

[ivoryec]

I've read that when you give MACS an IP file and an input file (-t and -c), the larger library is scaled to the smaller library.

My question is: Do the bdg files (pileup and lamda) reflect this scaling?

I have samples from different conditions and I would like to show the graphs side by side but the library sizes were quite different. Each sample has an IP and an input file, so each sample was run through MACS independently.
My understanding is that each pileup can be considered to be normalized for library size in comparison to the corresponding lambda, but not between different IP samples.... is that correct?

[ivoryec]

I posted this question to MACS github issues and received this response.

Answer 1

[Manvendra Singh]

I guess

No, Its not normalized by library size but its background corrected file, but you can normalize it afterwards, if you are doing statistics on them.(just divide the average tag count by number of reads mapped in million)

[ivoryec]

I didn't think the pileup graph took into account the background (input) sample. When I compare them, their ratio at the summits is almost exactly equal to the fold enrichment. I thought I would have to integrate the two graphs (pileup and lambda) to get a background corrected file.

I was considering making a new graph of the ratio between the pileup and the lambda for each sample and using that to visualize comparisons between the samples. Would that be wrong?

[Manvendra Singh]

pileup is shifted signal, you can divide your window into smaller bins, then count the number of tags in each window.

I am not sure about ratio, that it would tell you anything, for this you can make a threshold and just look into peaks

[cthangav]

Would you be able to tell me how the pileup value in a MACS2 atac-seq output file is calculated? Is it the number of counts at the peak summit? I've been looking for the specific definition so if there is a manual or resource that explains this value that would be very helpful.