Hi I am working with a chip which contains "empty" spots, i.e. nothing has been spotted there, "landing lights", and actual spots "cDNA". I would like to exclude "empty" and "landing light" spots from my dataset before normalisation. So, this works fine: library(limma) targets=readTargets(...) spottypes=readSpotTypes(...) files = c(targets$FileNameCy3, targets$FileNameCy5) numrows = length(files)/2 dim(files) = c(numrows,2) rawdata = read.imagene(files, path=...) g2=data.frame(ID=rawdata$genes$"Gene ID",Name=rawdata$genes$"Gene ID") status = controlStatus(spottypes,g2) normdata = normalizeWithinArrays(rawdata) ... and I tried this to exclude the "empty" spots and "landing lights": raw2=rawdata[status=="cDNA",] normdata = normalizeWithinArrays(raw2,method="none") ... which works fine, but with normdata = normalizeWithinArrays(raw2) I get this >Error: subscript out of bounds Any suggestions? Thanks Thomas _________________________ Thomas Schlitt British Antarctic Survey th_schlitt@gmx.de -- +++ Jetzt WLAN-Router für alle DSL-Einsteiger und Wechsler +++ GMX DSL-Powertarife zudem 3 Monate gratis* http://www.gmx.net/dsl

Hi Thomas, > Hi > I am working with a chip which contains "empty" spots, i.e. nothing has > been spotted there, "landing lights", and actual spots "cDNA". I would > like to exclude "empty" and "landing light" spots from my dataset before > normalisation. I think it is more common to exclude these sorts of spots after normalization (but before fitting a linear model). > normdata = normalizeWithinArrays(raw2) > > I get this >>Error: subscript out of bounds normalizeWithinArrays uses print-tip-group loess normalization by default. Because you did not specify the printer layout as an argument of nomalizeWithinArrays(raw2), the layout is expected to be stored in: raw2$printer. The normalizeWithinArrays function may expect your total number of genes to be the product of the components in the printer layout, i.e. ngrid.r x ngrid.c x nspot.r x nspot.c which is not true if you have excluded spots. Hope this helps, James

> Hi > I am working with a chip which contains "empty" spots, i.e. nothing has > been > spotted there, "landing lights", and actual spots "cDNA". I would like to > exclude "empty" and "landing light" spots from my dataset before > normalisation. > > So, this works fine: > library(limma) > targets=readTargets(...) > spottypes=readSpotTypes(...) > files = c(targets$FileNameCy3, targets$FileNameCy5) > numrows = length(files)/2 > dim(files) = c(numrows,2) > rawdata = read.imagene(files, path=...) > g2=data.frame(ID=rawdata$genes$"Gene ID",Name=rawdata$genes$"Gene ID") > status = controlStatus(spottypes,g2) > normdata = normalizeWithinArrays(rawdata) > > ... and I tried this to exclude the "empty" spots and "landing lights": > > raw2=rawdata[status=="cDNA",] > normdata = normalizeWithinArrays(raw2,method="none") > > ... which works fine, but with > > normdata = normalizeWithinArrays(raw2) Use instead w <- as.numeric(status=="cDNA") normdata <- normalizeWithinArrays(rawdata,weights=w) Remember that limma expects to get complete arrays if you wish to use a print-tip based normalization method. Therefore keep all the spots in your data object but give zero weight to the spots you don't want to have any influence on the normalization. Gordon > I get this >>Error: subscript out of bounds > > Any suggestions? > > Thanks > Thomas > > _________________________ > Thomas Schlitt > British Antarctic Survey > th_schlitt@gmx.de