Priscila Grynberg <priscilag at="" ...=""> writes: > > Dear Gordon, > > Thanks for the explanation. I didnt realize robust spline was dependent of > print-tips. > > Sincerely, > > Priscila > Dear all, I also applied the robust spline with the Agilent two colour array data, however, I did not get any errors.... are the error messages depend on the package that I use? This is what I did: targets <- readTargets(file = cfg$target.files); RG <- read.maimages( files = targets$FileName, source = "agilent", columns = list(G = "gMedianSignal", Gb = "gBGMedianSignal", R = "rMedianSignal", Rb = "rBGMedianSignal"), annotation = c("Row", "Col", "FeatureNum", "ControlType", "ProbeName", "GeneName") ); RG <- backgroundCorrect(RG, method="minimum", offset=1) MA <- normalizeWithinArrays(RG, method="robustspline") ### Session Info ###################################################################### ########## ### R version 2.14.2 (2012-02-29) Platform: x86_64-unknown-linux-gnu (64-bit) locale: [1] C attached base packages: [1] splines grid stats graphics grDevices datasets methods [8] utils base other attached packages: MASS_7.3-17 latticeExtra_0.6-19 lattice_0.20-0 limma_3.10.3 > On Thursday, March 8, 2012, Gordon K Smyth <smyth at="" ...=""> wrote: > > Dear Priscila, > > > > You can't apply robust-spline normalization to an Agilent array. > Robust-spline in limma is intended to moderate the loess curves over > different print-tip groups. But Agilent arrays don't have print- tips. For > an Agilent arrays, only global loess normalization is meaningful, and that > does not require the layout. > > > > Best wishes > > Gordon > > > >> Date: Wed, 7 Mar 2012 09:16:22 -0300 > >> From: Priscila Grynberg <priscilag at="" ...=""> > >> To: <bioconductor at="" ...=""> > >> Subject: [BioC] RobustSpline normalization method applied to agilent > >> data > >> > >> Dear BioCs, > >> > >> I received some agilent data to analyse using Limma, and I'm facing a > >> problem when I tried to apply robustspline normalization within arrays > >> method. > >> > >> ##Reading the files: > >> > >> RG = read.maimages(targets,,source="agilent", columns = list(G = > >> "gMedianSignal", Gb = "gBGMedianSignal", R = "rMedianSignal", Rb = > >> "rBGMedianSignal"), annotation = c("Row", "Col","FeatureNum", > >> "ControlType","ProbeName","SystematicName")) > >> > >> ##Background correction: > >> > >> RGn <- backgroundCorrect (RG, method="normexp", offset=50) > >> > >> #Normalization step > >> > >> MA <- normalizeWithinArrays(RGn, method="robustspline") > >> > >> Error in normalizeWithinArrays(RGn, method = "robustspline") : Layout > >> argument not specified > >> > >> ##Setting the printer information: > >> > >> nr <- length(unique(RG$genes$Row)) > >> > >> nc <- length(unique(RG$genes$Col)) > >> > >> RG$printer <- list(ngrid.r=1,ngrid.c=1,nspot.r=nr,nspot.c=nc) > >> > >> ## Trying again > >> > >> MA <- normalizeWithinArrays(RGn, RG$printer, method="robustspline") > >> > >> Error in X[O, ] <- ns(A[O], df = df, intercept = TRUE) : number of items > to > >> replace is not a multiple of replacement length > >> > >> I don't have the gal file like for this agilent slides. Can this be > exactly > >> the problem? Do you have any sugestions? > >> > >> > >> Cheers, > >> > >> Priscila > >> > >> > >> -- > >> Priscila Grynberg, D.Sc.- Bioinformatics > >> Laborat?rio de Gen?tica Bioqu?mica > >> Universidade Federal de Minas Gerais > >> Tel: +55 31 3409-2628 > >> CV: http://lattes.cnpq.br/8808643075395963 > > > > ______________________________________________________________________ > > The information in this email is confidential and inte...{{dropped:12}} > > > > _______________________________________________ > Bioconductor mailing list > Bioconductor at ... > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor

Dear Sylvia, My original motivation for creating the robustspline method was to moderate the normalization curves between multiple print tip groups. This was an important issue for the spotted two-colour microarrays that were popular ten years ago. With modern Agilent two colour arrays, there are no print tips and no print tip groups, so much of the motivation for robustspline normalization is no longer relevant. However the method can be used even when there are no print tips (or just one print tip group), in which case robustspline simply fits a single curve. In this situation, the method is closely analogous to global loess, with a regression spline replacing the loess curve and with high-breakdown robust regression replacing the loess robustifying weights. The method has potential advantages over loess normalization when there a lot of differential expression or the differential expression is assymetric. I do not actively promote it however. Priscilla got an error previously because she tried to set the printer layout herself and the settings did not agree with the number of probes on your arrays. If one specifies a single tip group with: RG$printer <- list(ngrid.r=1,ngrid.c=1,nspot.r=nr,nspot.c=nc) then nr*nc must equal the total number of probes on your arrays. It is usually best not to over-ride the settings that the software does itself unless you're very sure of what they are doing. I don't understand your question about error messages depending on which package is used, because robustspline normalization is something I created and is only available in the limma package. The software you are using is two Bioconductor releases out of date. Please update to current software. See the posting guide, which asks you to ensure that your packages are up-to-date. Is there any reason why you are setting the column names yourself instead of just using source="agilent.median"? Best wishes Gordon > Date: Tue, 27 Nov 2012 22:59:14 +0000 > From: sylvia <sylvia.shiah at="" oicr.on.ca=""> > To: <bioconductor at="" stat.math.ethz.ch=""> > Subject: Re: [BioC] RobustSpline normalization method applied to > agilent data > > Priscila Grynberg <priscilag at="" ...=""> writes: > >> >> Dear Gordon, >> >> Thanks for the explanation. I didnt realize robust spline was dependent of >> print-tips. >> >> Sincerely, >> >> Priscila >> > Dear all, > > I also applied the robust spline with the Agilent two colour array data, > however, I did not get any errors.... are the error messages depend on the > package that I use? > > This is what I did: > > targets <- readTargets(file = cfg$target.files); > > RG <- read.maimages( > files = targets$FileName, > source = "agilent", > columns = list(G = "gMedianSignal", Gb = "gBGMedianSignal", R = > "rMedianSignal", Rb = "rBGMedianSignal"), > annotation = c("Row", "Col", "FeatureNum", "ControlType", "ProbeName", > "GeneName") > ); > > RG <- backgroundCorrect(RG, method="minimum", offset=1) > MA <- normalizeWithinArrays(RG, method="robustspline") > > > ### Session Info > #################################################################### ############ > ### > R version 2.14.2 (2012-02-29) > Platform: x86_64-unknown-linux-gnu (64-bit) > > locale: > [1] C > > attached base packages: > [1] splines grid stats graphics grDevices datasets methods > [8] utils base > > other attached packages: > MASS_7.3-17 > latticeExtra_0.6-19 > lattice_0.20-0 > limma_3.10.3 > > > > > > > > > >> On Thursday, March 8, 2012, Gordon K Smyth <smyth at="" ...=""> wrote: >>> Dear Priscila, >>> >>> You can't apply robust-spline normalization to an Agilent array. >> Robust-spline in limma is intended to moderate the loess curves over >> different print-tip groups. But Agilent arrays don't have print- tips. For >> an Agilent arrays, only global loess normalization is meaningful, and that >> does not require the layout. >>> >>> Best wishes >>> Gordon >>> >>>> Date: Wed, 7 Mar 2012 09:16:22 -0300 >>>> From: Priscila Grynberg <priscilag at="" ...=""> >>>> To: <bioconductor at="" ...=""> >>>> Subject: [BioC] RobustSpline normalization method applied to agilent >>>> data >>>> >>>> Dear BioCs, >>>> >>>> I received some agilent data to analyse using Limma, and I'm facing a >>>> problem when I tried to apply robustspline normalization within arrays >>>> method. >>>> >>>> ##Reading the files: >>>> >>>> RG = read.maimages(targets,,source="agilent", columns = list(G = >>>> "gMedianSignal", Gb = "gBGMedianSignal", R = "rMedianSignal", Rb = >>>> "rBGMedianSignal"), annotation = c("Row", "Col","FeatureNum", >>>> "ControlType","ProbeName","SystematicName")) >>>> >>>> ##Background correction: >>>> >>>> RGn <- backgroundCorrect (RG, method="normexp", offset=50) >>>> >>>> #Normalization step >>>> >>>> MA <- normalizeWithinArrays(RGn, method="robustspline") >>>> >>>> Error in normalizeWithinArrays(RGn, method = "robustspline") : Layout >>>> argument not specified >>>> >>>> ##Setting the printer information: >>>> >>>> nr <- length(unique(RG$genes$Row)) >>>> >>>> nc <- length(unique(RG$genes$Col)) >>>> >>>> RG$printer <- list(ngrid.r=1,ngrid.c=1,nspot.r=nr,nspot.c=nc) >>>> >>>> ## Trying again >>>> >>>> MA <- normalizeWithinArrays(RGn, RG$printer, method="robustspline") >>>> >>>> Error in X[O, ] <- ns(A[O], df = df, intercept = TRUE) : number of >>>> items to replace is not a multiple of replacement length >>>> >>>> I don't have the gal file like for this agilent slides. Can this be >>>> exactly the problem? Do you have any sugestions? >>>> >>>> >>>> Cheers, >>>> >>>> Priscila >>>> >>>> >>>> -- >>>> Priscila Grynberg, D.Sc.- Bioinformatics >>>> Laborat?rio de Gen?tica Bioqu?mica >>>> Universidade Federal de Minas Gerais >>>> Tel: +55 31 3409-2628 >>>> CV: http://lattes.cnpq.br/8808643075395963 >>> ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}