Hi,

Being new to DESeq2 and differential expression analysis, I am a bit confused by the setting up of the design matrix of my experiment, as well as the downstream tests/contrasts.

I have:

- 3 genotypes : H, R and W. Being both exosome-related mutants, H and R are related. W is obviously wildtype.

- 3 timepoints : 0, 10 and 30 minutes.

- 2 conditions : treated and untreated. Timepoint 0 is untreated, while 10 and 30 are treated.

- 3 biological replicates per sample

Total = 24 libraries

Here is the design matrix I came up with:

genotype timepoint replicate condition exosome H_0_R1 H 0 1 U mut H_0_R2 H 0 2 U mut H_0_R3 H 0 3 U mut H_10_R1 H 10 1 T mut H_10_R2 H 10 2 T mut H_10_R3 H 10 3 T mut H_30_R1 H 30 1 T mut H_30_R2 H 30 2 T mut H_30_R3 H 30 3 T mut R_0_R1 R 0 1 U mut R_0_R2 R 0 2 U mut R_0_R3 R 0 3 U mut R_30_R1 R 30 1 T mut R_30_R2 R 30 2 T mut R_30_R3 R 30 3 T mut WT_0_R1 W 0 1 U ok WT_0_R2 W 0 2 U ok WT_0_R3 W 0 3 U ok WT_10_R1 W 10 1 T ok WT_10_R2 W 10 2 T ok WT_10_R3 W 10 3 T ok WT_30_R1 W 30 1 T ok WT_30_R2 W 30 2 T ok WT_30_R3 W 30 3 T ok

(As you can see, I don't have the R genotype at treated timepoint 10 (i.e. R_10_R1, R2 & R3).)

**My first question is:** do I need to include replicates as a factor? These are just biological replicates, not independent experiments, and timepoints are not paired (i.e. WT_0_R1 is not the same biological material as WT_10_R1, which would have been sampled twice). I believe not, but I'd like confirmation.

**Secondly, how should I write the design formula to answer questions such as:**

- Effect of being an exosome-mutant (independent of anything else) ?

- Effect of the treatment (independent of anything else) ?

- Effect of the timecourse ?

I am very confused about (most probably very basic) concepts such as : when I want to investigate the effect of being an exosome mutant, should the analysis account for all the other factors, or on the contrary all those other factors are not to be considered at all (given they will be found in both mutant and wt)?

Thanks in advance for any help!

That is much clearer to me now, thanks for the explanations!