Hello everyone! I’m working on an ATAC-seq project with four samples (no replicates), each sequenced to a different depth (e.g. 200 M, 300 M, 400 M paired-end reads, etc.). After marking duplicates on the BAMs, I’m generating BigWig tracks with deeptools bamCoverage using RPGC normalization (as recommended here: https://groups.google.com/g/deeptools/c/th96gaftAXQ). When I run computeMatrix and plot TSS enrichment (cluster of 5 genes), I still see fluctuations instead of a smooth curve that I haven’t been able to explain.
Could someone advise on:
- Whether RPGC is the best normalization strategy when you have no replicates but varying library sizes.
- How to calculate and apply the correct scale factors (e.g. using
the --scaleFactor option) if RPGC alone isn’t sufficient.
Any tips on achieving truly comparable BigWig tracks (and hence TSS plots) across samples would be hugely appreciated!
Thank you!
1 answer
See for code and discussion ATAC-seq sample normalization
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>> 100miomost will just be duplicates.