Hello all,

I am working on a RNA-Seq project that was sequenced without any biological replicates.

I know that it is bad practice to use this kind of data, but the researcher didn't seem to have a choice and this is the only data that i have to work with.

I have 4 samples, each one from a different condition, and i am interested in 4 comparisons with different conditions combinations.

I understood from the DESeq vignette that DESeq assumes that the samples that were entered to the analysis are all replicates, in order to estimate the dispersion. With that assumption, is it more accurate to build a data set and perform the normalization, estimations and testing separately for each comparison separately or should i still enter a raw counts table containing all 4 samples, estimate size factor and dispersions and then test each comparison separately?

With regular data with replicates, we usually normalize all samples together, but when assuming that different conditions are replicates, would it be more accurate to completely separate the comparisons?

Thank you very much,

Olga.

I will take this in consideration,

Thank you very much!