Im using bamCoverage on a file and ive set the bin size to 1 but also normalizing using RPKM for defined regions using a bedgraph file. The output from bamCoverage however is in binned segments each with a (RPKM) score. Am I correct in interpreting then that

1 - the output bedgraph tells me the (total) RPKM score for that particular bin?

2 - to get the total RPKM score for the region defined in the bedgraph I need to sum all the RPKM scores given for that particular region?

  • $\begingroup$ If I'm not mistaken, RPKM includes a "normalization" by unit of length, so it does not make sense to sum these values along a region. A weighted average makes more sense (were the weights would be the bin widths). $\endgroup$ – bli Dec 29 '20 at 8:36
  1. Yes
  2. The total RPKM for the region in the bedgraph file is the value already present in the bedgraph file. If you want to estimate the RPKM for a larger region composed of multiple bins then you can average the per-bin RPKM values. Note that adjacent bins with the same value are merged by deepTools, so you will need to account for this.

Note that RPKM is NOT a robust normalization and that I should remove it from deepTools so people stop using it.

  • 1
    $\begingroup$ @OP If you want a more robust normalization I suggest (disclaimer, I am the author of the post) biostars.org/p/413626/#414440 $\endgroup$ – ATpoint Dec 28 '20 at 16:52

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