Estimating Dispersions DESeq: ALL Conditions at once, or each comparison separately?

[carmeyeii]

Hi everyone,

I have an RNA-seq dataset which comprises 4 different conditions, and 2 biological replicates per condition, like so:

  cond1_1        cond1_2      cond2_1     cond2_2      cond3_1   cond3_2      cond4_1      cond4_2   
gene1       
gene2
gene3
gene4
gene5

Currently, I have been performing the sizeFactors function, as well as the estimateDispersions function on each table of 2 conditions (4 samples) at a time (the comparison in turn).

I make the data frame pertaining ONLY to comparison.X , then do estimateSizeFactors and estimateDispersions, and finally run the negative binomial Test on those results.

I am wondering, however, if it is best to supply DESeq with all the samples to estimateSizeFactors and estimateDispersions, and then run the paired-condition comparisons individually as usual. Might this provide more information per gene, or would it be counterproductive?

Thanks, Carmen

[Wolfgang Huber]

Dear Carmen
I am not sure it is possible to make a recommendation for one of the two possible choices you describe in general; this could depend on specific aspects of your experimental system or biological question. However - in your case, does it actually make a difference?
Best wishes
Wolfgang