Dear Mike, Thanks for explanations, I will follow your recommandations. Best regards Marie-Agn?s Le 31/08/2014 15:16, Michael Love a ?crit : > > Hi Marie-Agn?s, > On Aug 31, 2014 12:16 PM, "INRA - coutellec" > <marie-agnes.coutellec at="" rennes.inra.fr=""> <mailto:marie-agnes.coutellec at="" rennes.inra.fr="">> wrote: > > > > Dear all, > > > > My question is about the right design to use to perform LRT tests with > > DESeq2. I have two crossed factors, tra (2 levels) and pop (4 levels). I > > test the interaction term as follows: > > > > > ddsFullCountTable <- DESeqDataSetFromMatrix( > > > > + countData = countE, > > > > + colData = cda, > > > > + design = ~ pop*tra) > > > > > > > dds2inter.LRT <- DESeq(ddsFullCountTable, test="LRT", full=~pop*tra, > > reduced=~pop+tra) > > > res2<-results(dds2.LRT) > > > sum( res2$padj < 0.1, na.rm=TRUE ) > > [1] 389 > > > > and get 389 DEGs (out of 45249 contigs). Although this is very fine with > > me, I would like to know what exactly is listed in column > > 'log2foldchange' for this interaction term (change between what and > what?). > > See the help for ?results, where we describe the meaning of the LFC > column of a results object for test="LRT". > > The LFC is for just 1 of the 3 interaction terms (the last level of > both factors), while the p-value is for all interaction terms. > > As in linear models, an interaction term is an additional term to > account for differences in the tra effect across pop groups. It is an > additive term in the log2 transformed space, so an additional fold > change in the space of counts. > > > > > Next, I want to test for the "tra" effect, alone. I tried : > > > > > dds.LRT <- DESeq(ddsFullCountTable, test="LRT", full=~pop*tra, > > reduced=~pop+pop:tra) > > or: > > > dds.LRT <- DESeq(ddsFullCountTable, test="LRT", > > full=~pop+tra+pop:tra, reduced=~pop+pop:tra) > > > > and get the same error message: > > Error in nbinomLRT(object, full = full, reduced = reduced, betaPrior = > > betaPrior, : > > less than one degree of freedom, perhaps full and reduced models are > > not in the correct order > > > > whereas the command below works, although the two models differs by two > > elements (which shouldn't work in my view): > > > dds.LRT <- DESeq(ddsFullCountTable, test="LRT", > > full=~pop+tra+tra:pop, reduced=~pop) > > > > > > For the main 'tra' effect, you should just use the Wald test: > > results(dds, contrast=c("tra","B","A")) > > Filling in the two levels of tra. > > Or if this doesn't work (i don't know what version you are using), run > this first: > > dds = nbinomWaldTest(dds, betaPrior=FALSE) > > Mike > > > > > I understand it is somehow meaningless to eliminate a factor while > > keeping it through its interaction in a model. Thus, I plan to: > > 1) take out the 389 "interaction" DEGs > > 2) reestimate size factors and dispersion with an additive design > > (design=~pop+tra), from which I would be able to test the effect of > > "tra" and of "pop" very easily. > > > > Can anyone tell me if this is correct, or if I alternatively should keep > > "pop*tra" as design to estimate dispersion and then use "pop+tra" as > > full model and "pop" as reduced model in my LRT. > > > > Many thanks in advance, > > Marie-Agn?s > > > > -- > > Marie-Agn?s Coutellec > > INRA/Agrocampus-Ouest UMR0985 Ecology and Ecosystem Health > > Ecotoxicology and Quality of Aquatic Environments > > 65 rue de Saint-Brieuc > > 35042 Rennes cedex - FRANCE > > phone: +33 223 485 248 > > http://www6.rennes.inra.fr/ese_eng/ > > > > > > [[alternative HTML version deleted]] > > > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor at r-project.org <mailto:bioconductor at="" r-project.org=""> > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > -- Marie-Agn?s Coutellec INRA/Agrocampus-Ouest UMR0985 Ecology and Ecosystem Health Ecotoxicology and Quality of Aquatic Environments 65 rue de Saint-Brieuc 35042 Rennes cedex - FRANCE phone: +33 223 485 248 http://www6.rennes.inra.fr/ese_eng/ [[alternative HTML version deleted]]