Statistics (Opinion?) Question:
If you use FeatureCounts to calculate reads over some BED file (e.g., TSS coordinates) for ChIP-seq BAM files, is it best to normalize the BAM files to their inputs first, or is it better to allow downstream programs to perform their normalization on the raw counts? And for raw counts, what do you think is the best normalization to use: R/CPM, R/FPKM, TMM, TPM, or other (not so much for differential analysis, more like if you wanted to plot the normalized reads in a graph)? Again, specifically considering ChIP-seq and not RNA-seq in this case. I'm not a huge fan of downsampling so I wouldn't typically include that in the pipeline, but maybe you feel otherwise for normalization purposes?
Thanks! Looking forward to hearing your position.