Question: Design matrix (DESeq2)

Question

[ponganta]

Hey folks,

I want to ask whether I should conduct pairwise contrasts of my tissues in DESeq2, or whether I should test the tissue of interest against a background geneset.

I have tissue-specific counts from RNA-Seq data (triplicates). I want to analyse differential gene expression along a developmental gradient of one tissue type.

The tissues are well-separated in a PCA. Only the three stages of the query tissue are grouped together (albeit separated).

As background, we could use five additional tissues (reproductive and vegetative).

The design matrix for DESeq2 would thus look like the following:

ID	tissue	type	treatment
A1	A	vegetative	background
A2	A	vegetative	background
A3	A	vegetative	background
B1	B	vegetative	background
B2	B	vegetative	background
B3	B	vegetative	background
C1	C	reproductive	background
C2	C	reproductive	background
C3	C	reproductive	background
D1	D	reproductive	background
D2	D	reproductive	background
D3	D	reproductive	background
Qa1	Qa	query_stage_a	treatment_a
Qa2	Qa	query_stage_a	treatment_a
Qa3	Qa	query_stage_a	treatment_a
Qb1	Qb	query_stage_b	treatment_b
Qb2	Qb	query_stage_b	treatment_b
Qc1	Qc	query_stage_c	treatment_c
Qc2	Qc	query_stage_c	treatment_c
Qc3	Qc	query_stage_c	treatment_c

Which contrasts would you choose? Thanks for suggestions!

[ATpoint]

What does it mean 'test against a background geneset'?

[ponganta]

Sorry if my question is inappropriately worded. I want to analyse differential gene expression between the three different developmental stages of the query tissue. I want to take gene expression in the other tissues into account for that.

To this end, I first extracted results from the deseq-object as follows:

res <- results(deseq, 
    format = "DataFrame",
    contrast = c("tissue", "Qa", "Qb"),
    indepententFiltering = TRUE,
    alpha = 0.5,
    pAdjustMethod = "BH")

In essence, I'm doing pairwise analyses of developmental stages. I wanted to check if my analysis is appropriate as-is. Thanks for asking!

[Shred]

Update your question title using more specific terms and be more concise on which is your biological question. As it is, your experiment isn't in triplicate (Qb has only 2 replicates).