Question: DiffBind with highly variable FRiP (Fraction Reads in Peaks)
gravatar for GFM
3 months ago by
European Union
GFM20 wrote:

Hello, I am analyzing H3K27Ac ChIP seq samples from paired samples from disease and healthy tissues (each pair from one individual).
Peaks were called with MACS2. The number of peaks and the FRiP (fraction of peaks in reads) is very different between the two experimental groups: IPsample NumberPeaks FRiP Disease1 73186 11 Disease2 56963 6.3 Disease3 106123 18 Disease4 58564 8.3 Normal1 18400 2 Normal2 25585 2.9 Normal3 37038 3.6 Normal4 24459 2.8

I don't know whether the difference is due to real biology, or due to some technical problem. I used DiffBind for detecting differential H3K27Ac. As expected I get much more peaks which are up at the disease tissue. Are there any suggestions for what would be the best normalization in DiffBind in such a case, or how can I try understanding whether this is an artifact? Thank you

diffbind • 110 views
ADD COMMENTlink modified 10 weeks ago by Rory Stark2.8k • written 3 months ago by GFM20
Answer: DiffBind with highly variable FRiP (Fraction Reads in Peaks)
gravatar for Rory Stark
10 weeks ago by
Rory Stark2.8k
CRUK, Cambridge, UK
Rory Stark2.8k wrote:

If you have a prior expectation of a unidirectional shift in signal intensity toward one sample group (the disease condition), you should avoid normalizing use an RNA-seq method that assumes that there is a core of sites that don't change, and that changes will occur in both directions.

The default methods used by DiffBind should work, namely method=DBA_DESEQ2 and bFullLibrarySize=TRUE. Thhis will result in normalizing only for sequencing depth (total reads) for each sample.

ADD COMMENTlink written 10 weeks ago by Rory Stark2.8k
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