Hi,

I am trying to understand the DESeq2 design formula.  I am new to RNAseq, new to DESeq2 and new to writing design formulas/matrix (not a statistician or bioinformatician). It may be a very basic level question. But I will really appreciate if someone can help me understand.

I am using counts from RSEM and used tximport for data import.

My experiments has two groups (low temperature and high temperature). I have two technical replicates for each group and 6 biological replicates from each technical replicate.

I used 

sampletable <- data.frame (temperature= factor (rep(c("low", "high"), each = 12)

But in this design it did not take into account the technical replicates. Could anyone help me to understand how I include the technical replicate, So that I can confirm that the result is not an effect of the technical replicates (for any reason) but the effect of the temperature.

Thank you so much for the help in advance.

The recommendation is to collapse technical replicates, as these don't give information about the biological variability that is used in testing across groups of samples. Have you seen the collapseReplicates() function in the vignette?