**290**wrote:

Hi,

I only noticed this by chance, but it peaked my curiosity and couldn't find any documentation about it. I occasionally get asked about experiments where conditions are in singlet (no replication), and my typical mandate is to run the line of "no replication, no statistics", however in the case of making the most of data available, I was aware of DESeq2's single sample method in which all samples are treated as a single group for dispersion estimation. When running the same singlet design in Limma, I expected an error when testing the model fit, but I got nothing.

So my question is, what's Limma doing in the case of a singlet experiment *A-B, *as my assumption was that the moderated t-test couldn't be calculated without replication?

Some code just to make a reproducible example:

library(limma) set.seed(1234) pheno_table <- data.frame(Rep = c(paste0("A",1:8)), Variable = c("A","B","C","C","C","D","D","D")) mat_rows <- 100 matrix_in <- matrix(log2(rnorm(n = (mat_rows*nrow(pheno_table)), mean = 100, sd = 30)), nrow = mat_rows) design <- model.matrix(~0 + Variable, data = pheno_table) colnames(design) <- gsub("Variable", "", colnames(design)) contrasts <- c("A_Vs_B" = "B-A") contrast_mat <- makeContrasts(contrasts = contrasts, levels = colnames(design)) colnames(contrast_mat) <- names(contrasts) fit <- eBayes(contrasts.fit(lmFit(matrix_in, design),contrast_mat)) topTable(fit, coef = 1, number = 5, p.value = 0.1) # logFC AveExpr t P.Value adj.P.Val B # 92 -2.909594 6.21836 -4.935319 0.0009739746 0.09739746 -0.3183399

**32k**• written 4 months ago by andrew.j.skelton73 •

**290**