Question: Design matrix (DESeq2)
Entering edit mode
13 months ago
ponganta ▴ 590

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!

RNA-Seq DESeq2 • 742 views
Entering edit mode

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

Entering edit mode

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!

Entering edit mode

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).


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