Hello Everyone,

I use BEDtools extensively for my data analyses and I have a confusion about the BEDtools coverage tool.

here is a summary:

Aim of exercise: to compute the % genic length targeted in my chIP (over Input).

1. bin my metagenes of interest (metagenes.bed) into consecutive bins of 100bp size using BEDtools windowMaker
2. compute number of reads over the gene bins for IP.bam and Input.bam respectively using BEDtools coverageBED
3. in the output of coverageBED, normalize the number of reads per bin in IP & Input by CPM calculation
4. calculate log2ratio of normalized IP/Input to see which gene bins have log2ratio >=1
5. sum up the basepairs across gene bins which have log2 ratio >=1
6. this summed up value should be the total genic length that is enriched in my IP.

or not?

I have done all these 6 steps. My question is this:

Before calculating the log2ratio (IP/INPUT), I normalise by CPM as mentioned above. BUT, do I also have to normalize by "breadth of coverage" per bin that is reported in the output of coverageBED?

If yes, how do I do this? This will significantly affect my final log2ratios and the total base pairs which I will consider as my genic length enriched in my IP.

Thank you so much in advance for your help.