Hello!

I have recently started to use Deeptools for my ATAC-seq data analysis and would like to get some feedback on the conversion of the BAM files to bigwig coverage tracks. I am interested in looking at TSS metaprofiles using either all fragments or fragments of only certain sizes.

For an all-inclusive coverage:

bamCoverage --bam in.bam --outFileName out.bw --binSize 1 --normalizeUsing RPGC --effectiveGenomeSize x

Am I correct in normalizing to 1x coverage? From what I have seen in the literature, this is what most people are doing with ATAC-seq data. Also, I am uncertain about the effective genome size, which depends on read length. I have trimmed the Tn5 adapter sequence prior to alignment and have now varying read lengths.

For nucleosome-free regions:

bamCoverage --bam in.bam --outFileName out.bw --binSize 1 --normalizeUsing RPGC --effectiveGenomeSize x --skipNonCoveredRegions --extendReads --maxFragmentLength 120

I have seen that some people also use --minFragmentLength 40, but it doesn't seem to have a great effect when I look at the metaprofiles or in the genome browser. What is your experience? Also, what are your thoughts on skipping non-covered regions and extending the reads to full fragments? Especially the latter confuses me quite a bit. Does the extension result in the entire fragment being counted? If I don't specify this option, is only the 5' read end counted? I am not quite understanding which part of the read is counted by the tool. Shouldn't each read be only counted once instead of throughout its length?

For mononucleosome fragments:

bamCoverage --bam in.bam --outFileName out.bw --binSize 1 --normalizeUsing RPGC --effectiveGenomeSize x --MNase

In this specific case, people seem to not skip non-covered regions - why is that? In my understanding the above code creates files where only the center (3 bp) of fragments between 130 and 200 bp is considered. This reveals the position of the nucleosomes, correct? What about focusing on Tn5 cut-sites? Would I need to use --Offset 1? How exactly?

These are quite a lot of questions. It's quite hard for me to judge what is commonly used for those specific purposes mentioned above. Thanks to anyone who has taken the time to read this and I appreciate all input and help!