When an MDS plot is made using plotMDS.DGEList(), if the DGEList object that is supplied as an argument contains normalization factors, are they used to make the MDS plot? I'm told that they are, and yet when I look at the function code, it appears that unnormalized raw counts are being internally normalized in the function without using the normalization factors present in the DGEList object. I'm pasting the relevant code from plotMDS.DGEList() below. I don't see any use of x$samples$lib.size or x$samples$norm.factors, which contain the normalization factors, within the function. -- output of sessionInfo(): subdata <- x$counts[o, ] gm <- function(x) exp(mean(log(x))) myFun <- function(delta, y, ...) colSums(condLogLikDerDelta(y, delta, ...)) for (i in 2:(nsamples)) { for (j in 1:(i - 1)) { mm <- subdata[, c(i, j)] rs5 <- rowSums(mm) > 5 norm <- t(t(mm)/colSums(mm)) * gm(colSums(mm)) delta <- optimize(myFun, interval = c(1e-04, 0.99), tol = 1e-06, maximum = TRUE, y = norm[rs5, ], der = 0, doSum = FALSE) dd[i, j] = sqrt(delta$maximum/(1 - delta$maximum)) } } -- Sent via the guest posting facility at bioconductor.org.

Dear Ted, >From the code you give, you don't seem to be using the latest release of Bioconductor, which was released on 3 October 2012, see: http://www.bioconductor.org/news/bioc_2_11_release/ Type sessionInfo() to confirm what packages you are using. The current version of edgeR is 3.0.3. Nevertheless, I think your point is correct. plotMDS.DGEList is using norm.factors in part of the code (when it calls estimateTagwiseDisp) but not everywhere. I have committed a fix to edgeR today in edgeR 3.0.3 to make sure that plotMDS.DGEList consistently uses norm.factors. To see the fix you will need to install edgeR from Bioc 2.11. It may take a couple of days to become available. Best wishes Gordon > Date: Fri, 9 Nov 2012 15:51:52 -0800 (PST) > From: "Ted Toal [guest]" <guest at="" bioconductor.org=""> > To: bioconductor at r-project.org, twtoal at ucdavis.edu > Subject: [BioC] edgeR plotMDS.DGEList() use of normalized counts > > > When an MDS plot is made using plotMDS.DGEList(), if the DGEList object > that is supplied as an argument contains normalization factors, are they > used to make the MDS plot? I'm told that they are, and yet when I look > at the function code, it appears that unnormalized raw counts are being > internally normalized in the function without using the normalization > factors present in the DGEList object. I'm pasting the relevant code > from plotMDS.DGEList() below. I don't see any use of x$samples$lib.size > or x$samples$norm.factors, which contain the normalization factors, > within the function. > > > -- output of sessionInfo(): > > subdata <- x$counts[o, ] > gm <- function(x) exp(mean(log(x))) > myFun <- function(delta, y, ...) colSums(condLogLikDerDelta(y, > delta, ...)) > for (i in 2:(nsamples)) { > for (j in 1:(i - 1)) { > mm <- subdata[, c(i, j)] > rs5 <- rowSums(mm) > 5 > norm <- t(t(mm)/colSums(mm)) * gm(colSums(mm)) > delta <- optimize(myFun, interval = c(1e-04, 0.99), > tol = 1e-06, maximum = TRUE, y = norm[rs5, ], > der = 0, doSum = FALSE) > dd[i, j] = sqrt(delta$maximum/(1 - delta$maximum)) > } > } > > ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}