I have a question about testing main effects in a multiple-factor design with interaction term and another one on how to use the fdrtool. I have used it in the following way and I would like to know if this is correct or if it should be done differently.
In my case, I have RNAseq data from an experiment in which I reciprocally infected hosts with parasites from two sources. The design is full rank and I have three replicates per experimental host-parasite combination.
dds <- DESeqDataSetFromMatrix(countData = countData, colData = colData, design = ~ Host_origin + Parasite_origin + Host_origin:Parasite_origin) dds <- DESeq(dds) res <- results(dds, pAdjustMethod = "BH") res summary(res) resultsNames(dds) Host = results(dds, contrast=c("Host_origin","1","2")) Host2 <- Host[ !is.na(Host$padj), ] Host2 <- Host2[, -which(names(Host2) == "padj")] FDR.Host2 <- fdrtool(Host2$stat, statistic = "normal", plot = T) FDR.Host2$param[1, "sd"] Host2[,"padj"] <- p.adjust(FDR.Host2$pval, method = "BH") par(mfrow=c(1,2)) hist(Host$pvalue, col="lavender") hist(FDR.Host2$pval, col = "royalblue4", main = "WT vs Deletion, correct null model", xlab = "CORRECTED p-values") res_Host_sig = subset(Host2, padj<0.05 & abs(log2FoldChange)>=1.5)
I repeated the FDR correction steps for the main factor Parasite_origin and for the interaction term Host_origin:Parasite_origin. In each of the cases, the histogram with corrected p-values looks better.
I am interested in which contigs are up/down regulated for the main factors, host origin and parasite origin. In an ANOVA, if the interaction term is not significant, one would leave out the interaction term and test the new model without interaction. Does the results function in DESeq2 automatically do that? If not, is it possible to say anything about significantly up/down-regulated contigs depending on the main factors in a multiple factor design, and how?
My second question is about the FDR correction that I have done. Is it fine to do an FDR correction on each of the result contrasts or should an FDR correction be done only once for the overall dataset, after which the results should be extracted? If the FDR correction should only be done once (overall), how can I get the wanted contrasts for the different main effects and interaction?