normalization of POL2 ChIP-seq for the calculation of POL2 Pausing Ratios

Question

[Bogdan]

Dear all,

please could you advise about a way (ie a package/function in BioC) to normalize the ChIP-seq data for the calculation of POLYMERASE2 PAUSING RATIO (PR) for all the genes in the genome ;

we have a POL2 ChIP-seq dataset, and we compute the PAUSING RATIO (PR) as the ratio between the regions :

a. -- the read density in a region (-100,+300) around the TSS

b. -- the read density in a region (+1kb,+5kb) downstream of TSS

I know that we could normalize in edgeR or DESEq2 the ChIP-seq data;

another question being, shall we normalize the read counts in each region a) and b) above :

-- separately, -- together, or -- would it be legitimate to directly normalize the PR (Pausing Ratios) ?

thanks a lot,

bogdan

Answer 1

[Carlo Yague]

If you mean normalizing for library size, then you don't need to. That is because the ratio a/b does not depend on library size. In any case, you should not normalized a and b separately. On a side note, you should also be careful when both a and b are small, this could lead to wrongly huge ratio.

[Bogdan]

Thank you very much, Carlo ;)