Question: Removing GC content bias and trended bias from ChIP-seq data
gravatar for James Ashmore
3.8 years ago by
James Ashmore3.0k
UK/Edinburgh/MRC Centre for Regenerative Medicine
James Ashmore3.0k wrote:

Note: Cross-posted to Biconductor support site

I am doing DB analysis of ChIP-seq data using the csaw package. It seems I have a small trended bias and possible GC content bias in my data. Below are MA plots showing signal in the merged peaks using CPM normalisation (LHS) and Loess normalisation (RHS) to account for trended biases:

enter image description here

If I create MA plots coloured by GC content (showing only top/bottom 10% by GC content) there also appears to be a GC content bias (LHS) which can be fixed using CQN normalisation (RHS):

enter image description here

However, looking at the affect of CQN normalization (RHS) on all of the data, I'm not sure if it corrects the trended bias correctly (like the Loess normalization).

enter image description here

Also, when I look at the called differential peak regions in a genome browser using the CQN normalisation, some of the calls don't match what I can measure roughly by eye, suggesting the normalisation isn't appropriate. Both of these methods output an offset matrix which I supply to edgeR.

  1. Is there a way to combine the offsets produced by cqn and csaw to correct for both the trended bias and GC bias.
  2. Is there a better way to correct for these biases?
ADD COMMENTlink modified 3.8 years ago • written 3.8 years ago by James Ashmore3.0k

I suggest you to post it on Bioconductor supporting site as you are using csaw and edgeR. The authors of both softwares are very active there and you can quickly get professional answer.

ADD REPLYlink written 3.8 years ago by GZ1995380
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