I'm analyzing the RNAseq data using the RUVs tutorial. In this tutorial they use two functions,
uq <- betweenLaneNormalization(filtered, which = "upper") s <- RUVs(uq, 1:nrow(uq), k = 2, groups) design <- model.matrix(~x + s$W) y <- DGEList(counts = filtered, group = x) y <- calcNormFactors(y, method = "upperquartile") y <- estimateGLMCommonDisp(y, design) y <- estimateGLMTagwiseDisp(y, design) fit <- glmFit(y, design)
First question: Can I use the "median" instead of "upper" in betweenLaneNormalisation because it is giving me a RLE plot better than "upper"?
Second question: If I'm using "median" in above question then is it appropriate to calculate the normalization factor using the "upperquartile"method? If not then what is better option?