Hi,

I'm having issue with dba.contrast() in DiffBind v3.4.0.

I have two conditions, and each condition has 3 replicates. I'm working a simple differential peak analysis between treatment vs. control. Also, in my sampleSheet, my treatment samples come first then the control samples are under, because I wanted the Fold to be log2(treatment - control)

My issue is that I find the results to be inconsistent when I change the settings of the contrast, and I don't know which method is valid.

First approach is the automatic way:

  DBdata$contrasts=NULL
  DBdata <- dba.contrast(DBdata, categories = DBA_CONDITION) 
  DBdata <- dba.analyze(DBdata, method=DBA_ALL_METHODS, bParallel = TRUE)
  dba.show(DBdata, bContrasts=TRUE)

I ended up with 16,851 diffPeaks. The fold is log2(control - treatment), but I used bFlip to get what I want.

However, with the second approach, which is the following:

  DBdata$contrasts=NULL
  DBdata <- dba.contrast(DBdata, design=FALSE, group1=DBdata$masks$treat, group2 = DBdata$masks$untrt, name1="treat", name2="untrt")
  DBdata <- dba.analyze(DBdata, method=DBA_ALL_METHODS, bParallel = TRUE)
  dba.show(DBdata, bContrasts=TRUE)

While the order is fixed log2(treatment - control), I get 24,165 diffPeaks.

I don't know why the results are not consistent, and I hope you can help me to figure out which one is the right setting that I can trust..

Thank you

The first way, where you do not say design=FALSE, is preferred. The second way is included for backward compatibility, yielding the results DiffBind would give prior to version 3, but does not follow current best practices.

You can switch which condition is used as the reference using the reorderMeta parameter:

 DBdata <- dba.contrast(DBdata, categories = DBA_CONDITION,
                        reorderMeta = list(Condition="untrt"))