Hi there. I am currently trying to use DESeq2 analysis on some samples, but this is my first time using this, so I am looking at a lot of videos and examples, but I still don't quite understand how to write the R-script/code for what it is I want to do. I will try and give a detailed layout of my study.

I am studying the effect of a tomato virus on 2 plant lines, Resistant and susceptible. For each plant line I have 2 groups, infected and control, and I have 2 time points, 15 days post infection and 35 days post infection (dpi), and this also counts for the control group (they were inoculated with an empty plasmid to account for biological reactions to plant damage as well, to keep as many factors constant).

Now, I want to see how the genes were differentially expressed:

• for each time point of each plant line (15dpi vs control and so on)
• between the time points within each plant line (15dpi vs 35 dpi)
• between the two plant lines (15dpi resistant vs 15dpi susceptible and 35dpi resistant vs 35dpi susceptible)

Now I know that this means I am looking at different levels. But now my question is, do I use the contrast() or how do I approach this.

Here is my sample information: (3 biological replicates for control, 4 biological replicates for infected)

Resistant
Sample  Treatment   Time point  Plant Line
RVR1:15 Infected    15  Resistant
RVR2:15 Infected    15  Resistant
RVR3:15 Infected    15  Resistant
RVR4:15 Infected    15  Resistant
RVR1:35 Infected    35  Resistant
RVR2:35 Infected    35  Resistant
RVR3:35 Infected    35  Resistant
RVR4:35 Infected    35  Resistant
RCR1:15 Control         15  Resistant
RCR2:15 Control         15  Resistant
RCR3:15 Control         15  Resistant
RCR1:35 Control         35  Resistant
RCR2:35 Control         35  Resistant
RCR3:35 Control         35  Resistant

Susceptible
Sample  Treatment   Time point  Plant Line
SVR1:15 Infected    15  Susceptible
SVR2:15 Infected    15  Susceptible
SVR3:15 Infected    15  Susceptible
SVR4:15 Infected    15  Susceptible
SVR1:35 Infected    35  Susceptible
SVR2:35 Infected    35  Susceptible
SVR3:35 Infected    35  Susceptible
SVR4:35 Infected    35  Susceptible
SCR1:15 Control         15  Susceptible
SCR2:15 Control         15  Susceptible
SCR3:15 Control         15  Susceptible
SCR1:35 Control         35  Susceptible
SCR2:35 Control         35  Susceptible
SCR3:35 Control         35  Susceptible

Can I please ask for some assistance on this matter, I will greatly appreciate it.