I've read the DESeq2 documentation but I still don't understand how to analyze, interpret, and visualize multifactor designs for RNA-seq. Below is an example of what I am trying to figure out

Here, I have 4 groups that are differentiated by their values of controlVar and tx. I am interested in:

- How groups 2, 3, and 4 differ from group 1
- How groups 3 and 4 differ from each other
- How groups 3 and 4 differ from group 2

Any help on how to write the design formula to accomplish these would be tremendously appreciated. If I can provide further information, I will do so.

Here was my thought process:

- I'm guessing this looks at the effect of
`tx`

controlling for (i.e. stratifying by)`controlVar`

?`design = ~ controlVar + tx`

- I don't
*think*I am interested in the interaction of`controlVar`

and`tx`

because in theory the`controlVar`

should not be affecting anything - Would
`tx`

for groups 3 and 4 be considered a nested variable? If so, I don't know how to write this.

Thank you, but this does not help me with the nested variables issue. Basically, samples 4-12 have tx values that are nested within contralVar, and I'm not sure how to analyze this properly.