Calculate the total genie length targeted in my chIP

Question

[Zee_S]

Hello Everyone,

I have a list of annotated meta genes in a bed file. I also have my chIP.bam and INPUT.bam files. How do I calculate the total length of basepairs lying within these genes that are enriched for my chIP?

Thank you for your support!

Answer 1

[Devon Ryan]

You would need to call peaks and then see how much they overlap (e.g., with bedtools intersect or bedtools coverage).

[Zee_S]

Hello Devon,

Thanks a lot for your reply. I m looking at a broadly distributed mark so I don't call peaks. I usually compute a log2ratio of fold enrichment over input and use this to define my regions. Is it correct to say that using bedtools coverage, if I sum up the total number of bp per gene that have a log2ratio >=1.0 for example, is my enriched genic length ?

Many thanks

[Devon Ryan]

Possibly, though I would encourage you to use a domain caller for broad marks rather than using a simple log2 ratio.

[Zee_S]

Hello Devon, Thank you for your reply. Would you have any suggestion of a suitable way to get a log2 ratio when the input has zero reads assigned? Assuming I have comparable coverage in both IP and Input, can I assign a pseudo count of +1 to these "zero reads" bins? my worry is that this would change the ratios significantly and affect my downstream calculations of gene length.

even if I use a domain caller, as you recommend, this problem still persists (I think)

Thanks again.

[Devon Ryan]

As you surmised, you need to add a pseudo-count. I think we default to 1 for this in deepTools, I haven't seen any better suggestion for that value.