I am using deeptools to make bigwig files for 4 similar group of CHIPseq data. question1: my bigwig data are very different from each other, size difference some are around 259Mb but others are around 50Mb in size. What could cause this differences as my sample were treated the same and used the same way to sequence. I used RPKM to normalize. question2: when I am doing computeMatrix and plotHeatmap. The output of the plotprofile showing different basal levels. Is there a way to adjust the basel level or normalize it? Since I have already used RPKM to normalize the bigwig file, is there other normalization needed for this?
- the size difference is probably due to differences in genome coverage (the more genome regions that are covered with the reads, the larger the bigWig file), but they could also be due to using different bin sizes for the bigWig generation (e.g. a bin size of 10bp will generate larger files than a bin size of 1,000bp)
- normalizing for differences in background/enrichment efficiency is more cumbersome than one would hope for, Chapter 7 of the DiffBind vignettes covers the caveats quite exhaustingly. ATpoint has already written a great post about the issue and explains how he uses are more customized normalization scheme with deepTools