Dear all, I am analizyng Agilent G4121A mouse chips with LIMMA. My first three problems: 1) Layout: from the GAL file provided with the chip (and from the flat output file), I can only obtain the rows (105), and the columns (215). No information about the layout... is this due to the different technology (without printing tips)? If this is the case, I guess I don't need the printtiploess normalization. Is that correct? But what about the imageplot? How should I set the layout parameters in this function? 2) Is there a way (I am sure there is) to use the spot type information (stored in RG$genes$Status) also after the lmFit analysis (i.e. in the qqt plot and in the plot(A,M,pch=16,cex=0.1))? I would like to have the same colors for control spots also in these plots. 3) about the internal controls and duplicate spots on the Agilent arrays, their meaning is not clearly exposed in the accompanying documentation. I emailed the technical support, and they said that this information was developed with Rosetta (their analysis software), so it is not available to their customers... Don't you think this is a limitation of the analysis? Do you have more information about this? Thank you for your help ** hoping I did not post too much RTFM questions :-] ** Giovanni

At 08:39 AM 27/08/2004, Giovanni Coppola wrote: >Dear all, >I am analizyng Agilent G4121A mouse chips with LIMMA. >My first three problems: >1) Layout: from the GAL file provided with the chip (and from the flat >output file), I can only obtain the rows (105), and the columns (215). No >information about the layout... is this due to the different technology >(without printing tips)? Yes it is. Agilent doesn't have print tips. > If this is the case, I guess I don't need the >printtiploess normalization. Is that correct? I recommend method="loess". > But what about the imageplot? >How should I set the layout parameters in this function? Set ngrid.c=1, ngrid.r=1. BTW this will cause method="printtiploess" and method="loess" to be the same at the normalization step. >2) Is there a way (I am sure there is) to use the spot type information >(stored in RG$genes$Status) also after the lmFit analysis (i.e. in the qqt >plot and in the plot(A,M,pch=16,cex=0.1))? I would like to have the same >colors for control spots also in these plots. You can pass an argument col to qqt() or to plot(). This is a vector of integers or colour names ("red", "green" etc) of the same length as the number of points. E.g., plotcol <- RG$genes$Status plotcol[RG$genes$Status=="Control"] <- "red" plotcol[RG$genes$Status=="Gene"] <- "black" qqt(fit$t, col=plotcol) >3) about the internal controls and duplicate spots on the Agilent arrays, >their meaning is not clearly exposed in the accompanying documentation. I >emailed the technical support, and they said that this information was >developed with Rosetta (their analysis software), so it is not available to >their customers... Don't you think this is a limitation of the analysis? Do >you have more information about this? We haven't been able to get further information on the controls from Agilent either. It would certainly be nice to know they are but it doesn't limit the analysis. Many other arrays don't have these controls in the first place. Gordon >Thank you for your help >** hoping I did not post too much RTFM questions :-] ** > >Giovanni > >_______________________________________________ >Bioconductor mailing list >Bioconductor@stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor

At 08:39 AM 27/08/2004, Giovanni Coppola wrote: >Dear all, >I am analizyng Agilent G4121A mouse chips with LIMMA. >My first three problems: >1) Layout: from the GAL file provided with the chip (and from the flat >output file), I can only obtain the rows (105), and the columns (215). No >information about the layout... is this due to the different technology >(without printing tips)? If this is the case, I guess I don't need the >printtiploess normalization. Is that correct? But what about the imageplot? >How should I set the layout parameters in this function? You can't use imageplot() with Agilent arrays processed using AgilentFE software, because AgilentFE does not output rows for blank/empty spots while limma expects to get full arrays. You can use imageplot() with Agilent arrays processed by Genepix or similar. It is possible to get an image plot of Agilent/AgilentFE arrays using a combination of the limma and marray packages. The following code works for me: library(limma) RG <- read.maimages(fnames, source="agilent", path="AgilentFE") library(convert) y <- as(RG,"marrayRaw") nr <- max(RG$genes$Row) nc <- max(RG$genes$Col) maNgr(y) <- 1 maNgc(y) <- 1 maNsr(y) <- nr maNsc(y) <- nc included <- rep(FALSE,nr*nc) pos <- nc*(RG$genes$Row-1)+RG$genes$Col included[pos] <- TRUE maSub(y) <- included maImage(y,x="maRb") Gordon