On Thu, Dec 6, 2012 at 2:29 PM, Ryan C. Thompson <r...@thompsonclan.org> wrote: > Hi all, > > I'm working with an RNA-seq dataset where every biological sample has two > technical replicates. Is important for me to merge the technical replicates > so that my count matrix has exactly one column per biological sample, or is > it ok to leave them separate? My worry would be that leaving them separate > might make edgeR think that the technical replicates are biological > replicates, leading to an underestimate of dispersion and overestiamte of > significance. > > So does it matter whether I pool technical replicates (by simply adding > their counts together)? Is edgeR expected to give different results > depending on whether or not I pool?
Yes and yes. As you say yourself, some of your samples are technical replicates and some are biological. edgeR (and any other statistical packages) needs to know which is which, and you should be using a method that can differentiate between the two sources of variation. If you're using a package that cannot differentiate (and I am a bit out of touch, so I don't know if the current codebase of edgeR allows you to do so, but I am sure we will hear from people more familiar with the code), you are better off by summing the technical replicates. However, if properly used, tech reps allows you to quantify technical variability and biological variability which can be important (although underused in may bio papers). Remember that the software and statistical model does not magically figure out how your samples are related. Small pet peeve: there are many types of technical reps, from using the same library in different lanes of a flowcell (always uninteresting - or at least the interesting aspects of this level of variation has been addressed well in the literature - but very common), to actually doing two different library preps for the same rna sample (much more interesting, and people sometimes do this) to actually obtaining two different samples from the same tissue and person (almost never done, but by far the most interesting level of technical variation in my opinion). Best, Kasper > -Ryan Thompson > > _______________________________________________ > Bioc-devel@r-project.org mailing list > https://stat.ethz.ch/mailman/listinfo/bioc-devel _______________________________________________ Bioc-devel@r-project.org mailing list https://stat.ethz.ch/mailman/listinfo/bioc-devel
