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.