I have been told I cannot use WGCNA with only two conditions (control and treatment). I am not sure why that would be the case and I would like to confirm if this is true? I have 3 samples for each condition so a total of 6 samples and I am not correlating to a quantifiable trait, I am only correlating the modules to the conditions.
I believe it is not the condition but the number of replicates that are required for running WGCNA. Typically, you are required to have more than 15 samples. Ideally, I would recommend more than 30 replicates for each condition.
There is no correct or incorrect answer to this. To me, it makes more sense to perform WGCNA separately, so, one network for disease and one network for control:
Analysis 1:
generate network in Control
identify modules and hub genes
perform gene enrichment on the genes in each module
Analysis 2:
generate network in Disease
identify modules and hub genes
perform gene enrichment on the genes in each module
Analysis 3:
discuss differences between hub genes between Disease network and
Control network
discuss differences between modules (and their gene enrichments)
between Disease network and Control network
If you generate a single network for all samples (Disease + Control), then, after you identify modules, you can still try this: perform a linear regression between DiseaseStatus and the module eigenvalues:
Then, in this way, you would focus on the modules from which the regression produces p<0.05, as these modules may contain information about DiseaseStatus.