Question: Downsampling ChIP-seq BAM files with spike in normalization factor before feeding into differential binding analysis
gravatar for sikhtechai
14 months ago by
sikhtechai30 wrote:

I am planning to use exogenous chromatin as a spike in control with my actual sample from mouse to perform ChIP-seq for peak calling and differential binding analysis for histone modifications. this involves down-sampling the uniquely mapped read files to the calculated normalization factor from the spike in. This is seemingly helpful for peak calling and visualizing in genome browser. But I have not found any reference on whether it is considered in differential binding analysis. Since I am using Diffbind, I also could not find anything regarding this in the vignette. Could anyone please explain how this strategy might affect using the diffbind package? Or, does using spike-in for ChIP-seq normalization makes sense?

I greatly appreciate your time to read and answer to my question! Thank you in advance!

An example of this normalization process is given below(Image from Active motif's ChIP-seq spike in kit.) enter image description here

ADD COMMENTlink modified 14 months ago by nicolas.descostes130 • written 14 months ago by sikhtechai30

To my knowledge, DiffBind uses DESeq2 internally for differential analysis and normalization. DESeq2 expects raw counts and it is strongly recommended to provide these instead of customly normalized values.

ADD REPLYlink written 14 months ago by ATpoint17k

But technically, this will still be raw counts, just spike in adjusted for all samples. For RNA-seq, there are already a package (Ruvseq) which does this spike in normalization for the counts matrix, which can be used for DESeq2 analysis.

ADD REPLYlink written 14 months ago by sikhtechai30
gravatar for nicolas.descostes
14 months ago by
United States
nicolas.descostes130 wrote:

To my knowledge only csaw enables to include the spike-in before doing differential binding analysis with "searching". You can also have a look at the package that we are developing, ChIPSeqSpike:

If you know already where your histone modifications are located, I think that you can generate directly figures with our package. If you want to perform peak calling after scaling, you can use the "BamCoverage" function of deepTools by applying the scaling factors given by our "SpikeSummary" function.

We will integrate further solutions to perform all analysis at once in the near future.

ADD COMMENTlink written 14 months ago by nicolas.descostes130
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