Hi,

I am running a 3-factor analysis on RNA-Seq data. Plants have been inoculated with either bacteria or a mock solution; different timepoints after inoculation have been harvested and, finally, at every timepoint, two different tissues have been harvested: the directly inoculated tissue (“IN” tissue) and the surrounding tissue to this one (“OUT” tissue). Thus, the three factors are: time, inoculation and tissue.

This design provides loads of possible contrasts when going through edger and, hence, I’d like to simplify or to “compile” certain contrasts.  For instance: when looking at the interaction between tissue and time (at timepoint Tx as compared to T0), I might look at what happens when bacteria are inoculated OR when Mock solution is inoculated. Like this:

Tissue:Tx_Bact = ((Bact_IN.Tx – Bact_OUT.Tx) - (Bact_IN.T0 – Bact_OUT.T0))

Tissue:Tx_Mock = ((Mock_IN.Tx – Mock_OUT.Tx) - (Mock_IN.T0 – Mock_OUT.T0))

However, in order to simplify things, I thought we were not necessarily interested in the interaction when Mock itself is concerned, but only in comparison with the interaction when bacteria are inoculated.  In other words, I thought it’d be a good idea to subtract the Tissue:Tx_Mock interaction to the Tissue:Tx_Bact interaction, in order to get genes that are specific to the tissue:Tx interaction only when bacteria is inoculated. Like this:

Tissue:Tx_Compiled = ((Bact_IN.Tx – Bact_OUT.Tx) - (Bact_IN.T0 – Bact_OUT.T0)) - ((Mock_IN.Tx – Mock_OUT.Tx) - (Mock_IN.T0 – Mock_OUT.T0))

What do I get in summary with those contrasts?

• I only get Tissue:Tx_Mock DEGs at the first timepoint. This is OK, it makes sense in our system. Most of those genes (85% out of 554) are also Tissue:Tx_Bact at the same timepoint.  Interestingly though, I do not get Tissue:Tx_Compiled DEGs at the first timepoint.

• From the second timepoint on, I only get Tissue:Tx_Bact AND/OR Tissue:Tx_Compiled DEGs. At every timepoint I get ~1K Tissue:Tx_Compiled DEGs and ~5K Tissue:Tx_Bact DEGs. Also, 99% of the former are included within the latter.

Is my way of reasoning “interactions” as it should? Am I really looking with Tissue:Tx_Compiled at the interaction when bacteria are involved because I subtract the “Mock” inoculation?

I hope all this is clear enough. I thank you for any advice you could provide me with.

Best,

David

So if I understand your contrast correctly, you want to identify genes that respond differently in each tissue at a given time point, where that differential response is specific to the bacterial treatment, not the mock treatment. If so, I believe you have the correct contrast. Keep in mind, however, that a contrast involving so many groups inherits all the random noise from all of those groups, so your power to detect differential expression on this large contrast may be limited.

Another tool you can use to compare contrasts is limma's genas function, which evaluates the degree to which two contrasts are consistent with each other. This can help you determine the degree to which Tissue:Tx_Bact and Tissue:Tx_Mock are similar or different at a given time point. (Note that if you used edgeR for your differential expression analysis, you'll need to re-fit the same model with voom and limma to use genas.)