Hi all,

I'm analyzing atac-seq data with 4 genotypes and two biological replicate for each of them (8 samples overall). I used macs2 callpeak for peak calling and then used the .bam files and .narrowPeak files to apply Diffbind and get the binding matrix. Also, I'm working with DiffBind_2.14.0. I know that all my .narrowPeak files contain peaks in HOXa region (I annotated every single of them and hoxa regions overlap within all samples). However, I cannot find this region in the total binding matrix. I even set minOverlap=1 to get total number of unique peaks after merging overlapping ones, and then annotated all the regions but HOXa was not found.

So, I believe that DiffBind is filtering some of the regions somehow. My question is that what filtering steps the DiffBind is applying in order to generate the binding matrix?

dbaObj <-  dba(sampleSheet=cellSamples,peakCaller="narrow", 
     config=data.frame(doBlacklist=FALSE, doGreylist=FALSE, singleEnd=FALSE, yieldSize=5000000, 
                                            bUsePval=TRUE,th=0.01), scoreCol=5)

When I annotated all the regions from dba$merged I could find Skap2 but not HOXa. Below is a screenshot from uploading bw files to igv, showing the peaks for Skap2 and hoxa.
peaks from Skap2 vs. hoxa

Hoxa genes go from chr6:52,105,000-52,217,000, and this interval is available in all .narrowPeak files, but how come DiffBind remove it when making the binding matrix?

Many thanks,

Shiva.



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