Hi, I am new to DESeq2 and read through the previous questions, but still not quite sure how to deal with my data.   I have 3 replicates each of 14 different genotypes (each RNA batch made on a different day), and I would like to compare them all to wildtype and each other.  In my summarized experiment each sample is a row.  Should I try pooling the three replicates?  Also how do I generate the comparisons?  I didn't know what to use as my design...  Assuming contrast, but not sure how to call it.  my col_data has strain_name, mutant_allele and other non numeric information. Thankyou!

Alison 

It sounds like you should use ~batch + genotype. You can then make comparisons, after running DESeq(), using:

results(dds, contrast=c("genotype","geno1","WT"))

Or you can compare two genotypes with:

results(dds, contrast=c("genotype","geno2","geno1"))

You should organize your colData(dds) so it looks like:

batch genotype
    1       WT
    2       WT
    3       WT
    1    geno1
    2    geno1
    3    geno1
...

And you should set WT to be the reference level of genotype.