Hi Dr. Stark,

I was wondering if I could ask for your assistance with something DiffBind related please? My ChIP-seq experiment consists of five treatment groups (negative control, drug 1, drug 2, drug 3, drug 4) with two replicates per sample (i.e I have 10 .bam files in total, and 10 .narrowpeak files). Unfortunately, I do not have access to a high-performance computing cluster so am using the latest version of DiffBind in Galaxy for my analysis. For some reason the Galaxy version of DiffBind (2.10.0) does not allow you to enter more than two Conditions (i.e. I am only able to analyse negative control vs drug 1, or negative control vs drug 2, and am unable to analyze all five groups simultaneously). One way I have been able to partly get around this is to split the bam files up into two Conditions (Control vs Treated). However, the Galaxy version of DiffBind doesn't allow you to specify Tissue, Factor, and Treatment. Therefore, I am unable to separate out the four different drug-treated groups and carry out the comparisons I want.

Galaxy is able to export the R data from its DiffBind run, and I was wondering about the possibility of running DiffBind in R Studio on my computer and completing the analysis this way (after the resource intensive part has been completed in Galaxy). This may be a silly question, but is there a way for me to split the data up according to Tissue, Factor, Treatment etc. in the dBA objects so that I can undertake the comparisons that I want using Deseq2 in DiffBind?

I hope this makes sense and I would be very grateful for your input. Thanks!

I'm not involved with the Galaxy implementation but I see that it is quite out of date. More recent versions of DiffBind (starting with DiffBind_3.0) support arbitrarily complex GLM designs so you could model your experiment appropriately.

I suggest you have a look at the DiffBind vignette to get going with an R script. You will need to re-do the read counting step using dba.count() but even on a laptop this can be done in a reasonable amount of time for 10 bam files.