I have raw miRNA seq count data that I want to normalize using deseq normalization. I will export the data after normalization to conduct the downstream analysis (I will not use deseq2 package to conduct the differential analysis). In this case:
- Do I have to use the (vst) function or just use (counts, normalized=TRUE) and (normTransform, f=log2, pc=1)?
- Do I have to collapse the technical replicates using the (collapseReplicates) function before or after the deseq normalization?
- I also want to filter miRNAs based on a specific minimum number of counts in a minimum number of samples (e.g keep only those miRNAs with a minimum count of 10 in at least 30% of the samples). Do I have to do this filtering step before or after deseq normalization?