Greetings, I have been doing some RNA analysis with the limma package using the eBayes function. I have a matrix of counts consisting of 25322 genes and 23 samples. I intended to use the voom function to normalize my data. Here is my code and problem: design <- model.matrix(~factor(class)) colnames(test) <- c("Fasted", "Prefed") nf = calcNormFactors(sub, method = "TMM") voom.data <- voom(sub, design = design, lib.size = colSums(sub) * nf) voom.data$genes = rownames(sub) voom.fitlimma = lmFit(voom.data, design = design) voom.fitbayes = eBayes(voom.fitlimma) Warning message:In ebayes(fit = fit, proportion = proportion, stdev.coef.lim = stdev.coef.lim, : Estimation of var.prior failed - set to default value I tried looking into the error and couldn't find a clear answer in the archives (but I may have missed it). What I gather is one of the estimated parameters couldn't be and a default was entered instead. I don't think I should just fall on a default when there seems to be something that should be solvable. Curious I decided to do subsets of my data and the analysis would run up to 23800 rows but not 23801 and up. I then tried to use rows 10000:25322 and that works perfectly!?!? Is there some sort of limit on rows for eBayes? - I highly doubt it but I need to ask given what I am running into. Any thoughts would be sincerely appreciated as I am vexed by this. Thanks for your time, Sincerely, -- Charles Determan Integrated Biosciences PhD Student University of Minnesota [[alternative HTML version deleted]]