DESeq2 nested effect with 5 variables

Question

[sandeep.amberkar18]

Hello All,

I'm dealing with a rather complex RNAseq experiment that follows a nested structure of the tested variables that could be represented like this:

SampleName  tissue  temp    time    dev_stage   rep
Sample1 crown   21  am  ds1 rep1
Sample2 crown   21  am  ds1 rep3
Sample3 crown   21  am  ds1 rep4
Sample4 crown   21  am  ds2 rep1
Sample5 crown   21  am  ds2 rep2
Sample6 crown   21  am  ds2 rep3
Sample7 crown   21  am  ds2 rep4
Sample8 crown   21  am  ds3 rep1
Sample9 crown   21  am  ds3 rep2
Sample10 crown  21  am  ds3 rep3
Sample11 crown  21  am  ds3 rep4

From the several related posts related to experiment design on multifactor experiments, I came up with the design formula which looks like this:

exp.dds=DESeqDataSetFromMatrix(countData = counts_noZeros.df,
                               colData = exp_coldata,
                               design = ~0+tissue:temp:time:dev_stage)

I'm interested in determining the effect of these 4 variables on gene expression. For which I've 2 questions,

1) If I keep the design as is, is it correct that DESeq will account for a gene's expression considering a nested effect of these variables? In which case, is the model representation correct?

2) If I want to determine expression of only one of these variables should the design formula look something like this?

design = ~0+tissue

Any help is appreciated. With thanks.

Best, Sandeep

[ATpoint]

For these specific questions on DESeq2 I suggest you post this over at https://support.bioconductor.org/ to get expertise from the developer right away.

Answer 1

[leaodel]

Hi Sandeep. You have 3 identical variables in your design - tissue, temp and time - you won't be able to distinguish the contribution each variable, either in an 'isolated' or in a nested design, in the differential gene expression test. Even if you use one of these variables, you won't be able to infer that the genes are being DE are sole because of tissue as opposed to temp or time for example. Unfortunately, this is hard to solve.

[sandeep.amberkar18]

Hi Leaodel,

I'm sorry I should've been more clear about the exp. setup.

The experiment consists of:

• 2 Tissues -- Leaf, Crown
• 2 Temperatures -- 21, 27
• 2 Time points -- AM, PM
• 3 Dev Stages -- DS1, DS2, DS3

and 4 replicates for each.

I posted only a subset of the coldata matrix, as the full matrix would have been too much for a single post.