Hi all, 

I'm having problems with RNA-seq data. I have two replicates for five different treatments (all on the same cell line) and also two replicates for the control. I used DESeq2 to perform the differential expression analysis and I obtain that all the treatments are very similar to each other (I mean that the percentage of differentially expressed genes is always around 1 to 3% for each treatment). Only for one treatment I found that the percentage of differentially expressed genes is around 20%. This was unexpected and I can't find out a method to evaluate if it is a biological or a statistical issue. Do you have any idea on which analysis I could perform to confirm that I carried out correctly the differential expression analysis? If it is a biological issue do you have any idea of a possible explanation of such a substantial difference? 

Thanks,  
AriBo
> ...percentage of differentially expressed genes is always around 1 to
> 3% for each treatment). Only for one treatment I found that the
> percentage of differentially expressed genes is around 20%. This was
> unexpected...

What was unexpected about it? These numbers are not inconsistent with what we know is possible biologically. What is it that you know about your experiment, that leads you to believe this is an odd result? Given the information in your question there's nothing incompatible with a biological explanation. The PCA plot sets these samples apart from the others, so a large DE response could make sense. If you look at the values for DE genes and non-DE between each condition and control, does the data makes sense to you? Is there a core set of controls or housekeeping genes that exhibit similar behavior/characteristics across the experiment?

Is there anything about the biology in your results that does make sense? Or does NOT make sense (i.e. you applied heat shock, but your cells are exhibiting a cold shock response)? Drawing a line between each condition and at least one or a few genes one might expect to respond should be doable. Is there a confounding factor you haven't mentioned? For instance, was everything controlled for except condition? (i.e. all cells and conditions prepared and applied on the same day in the same laboratory by the same person?) or is there a batch effect whereby the outlier condition was applied 3 months later in a different laboratory on the other side of the world (but maybe nobody mentioned that)?