Hi all, 

was wondering if somebody could explain how limma produces genes that will be entered in to a venndiagram. I have produced a vennDiagram using limma results:

ebayesliversamples<-eBayes(contrastfitliversamples).......

resultsvennliversamples <- decideTests(ebayesliversamples, method="separate", adjust.method="BH", p.value=0.05)

then call vennDiagram to look at the result. This is really nice, however, I get a different number of genes when I then change the "include" argument. 

For "both" up-regulated and down-regulated genes, I get (just as an example) 168 genes on one outer ring (specific to one contrast).

Now, if I ask for genes that are up-regulated only (include="up") then the same outer ring as above now contains 301 genes. Similarly, include= "down" = 174 genes. 

This is really confusing for me, as I thought there should be 168 genes in total and then the up and down regulated genes would be a number out of the 168... 

My question is, why, when specifying to view genes that are specifically up-regulated or down-regulated, are there more genes than the total number of DE genes exclusive to this contrast when specifying "both"

Not sure of the code that might be useful here, I also couldn't directly attach the venn diagrams, however, I will respond with any requests for further code etc! But any help with this would be greatly appreciates as it is incredibly confusing!

Many thanks!

When you select 'both', the intersection can included genes that go up in one contrast and down in the other. If you select 'up' or 'down', the intersection will now only include those genes that go up or down in both contrasts, and those genes that were up in one contrast and down in the other end up in the unique sections of the diagram.

As an example, say you had 20 genes that were up in one contrast and down in the other. If you select 'both' then those 20 genes will appear in the intersection. If you select 'up' they will now appear in the unique section for the contrast in which they are up-regulated, and if you select 'down' they will appear in the unique section for the other contrast.