Hello Gordon and all, I am analyzing a RNA-seq experiment, and since I have quite some experience analyzing microarray data using linear models, I decided to go for the 'Voom - Limma' workflow. However, I do have some (practical) queries, and would appreciate feedback on these: - when transforming the counts data using voom(), is an addition normalization method recommended? E.g. cyclic loess? (normalize.method="cyclicloess"). Note that before applying Voom I already processed the count data with calcNormFactors(). - when applying the eBayes moderated statistical tests, is it still OK to use "trend=TRUE"? fit2 <- eBayes(fit,trend=TRUE). I am asking because voom() also estimates the mean-variance relationship in the data, and I don't want to 'over-correct'. BTW, in the plotSA() graphs I noticed only a minor effect of trend=TRUE. Thanks, Guido library(limma) samples <- read.delim("samples_descriptions.txt") counts <- read.delim("count_table_geneLevel_1703.txt", row.names=1) isexpr <- rowSums(cpm(counts)>1) >= 3 counts <- counts[isexpr,] #check: > dim(counts) [1] 10658 18; (62.1% of genes are retained) d <- DGEList(counts = counts, group = samples$Condition) d <- calcNormFactors(d) treatment <- as.factor(samples$Condition) design <- model.matrix(~0+diet) v <- voom(d,design,plot=TRUE) # defined contrast matrix, then: fit <- lmFit(v,design) fit1 <- contrasts.fit(fit,cont.matrix) fit2 <- eBayes(fit1,trend=TRUE) plotSA(fit2) topTable(fit2) > sessionInfo() R Under development (unstable) (2013-11-19 r64265) Platform: x86_64-w64-mingw32/x64 (64-bit) locale: [1] LC_COLLATE=English_United States.1252 [2] LC_CTYPE=English_United States.1252 [3] LC_MONETARY=English_United States.1252 [4] LC_NUMERIC=C [5] LC_TIME=English_United States.1252 attached base packages: [1] splines parallel stats graphics grDevices utils datasets [8] methods base other attached packages: [1] edgeR_3.5.28 multtest_2.19.2 Biobase_2.23.6 BiocGenerics_0.9.3 [5] limma_3.19.28 loaded via a namespace (and not attached): [1] MASS_7.3-29 stats4_3.1.0 survival_2.37-4 tools_3.1.0 > --------------------------------------------------------- Guido Hooiveld, PhD Nutrition, Metabolism & Genomics Group Division of Human Nutrition Wageningen University Biotechnion, Bomenweg 2 NL-6703 HD Wageningen the Netherlands tel: (+)31 317 485788 fax: (+)31 317 483342 email: guido.hooiveld@wur.nl internet: http://nutrigene.4t.com http://scholar.google.com/citations?user=qFHaMnoAAAAJ http://www.researcherid.com/rid/F-4912-2010 [[alternative HTML version deleted]]