Dear Pedro, Jim, and Bioconductor List, I received an error message when trying to normalize data with AgiMicroRMA > agiMicro.rma=rmaMicroRna(agiMicroraw, + normalize=TRUE, + background=FALSE) Error in if (min(dd.aux$Rb) < 0) { : missing value where TRUE/FALSE needed > agiMicro.rma=rmaMicroRna(agiMicroraw, + normalize=TRUE, + background=TRUE) Error in if (min(dd.aux$Rb) < 0) { : missing value where TRUE/FALSE needed > ###################################################################### ########## My session Inforrmation is: > sessionInfo() R version 2.11.0 (2010-04-22) i386-apple-darwin9.8.0 locale: [1] en_US.UTF-8/en_US.UTF-8/C/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] AgiMicroRna_1.2.0 preprocessCore_1.10.0 affy_1.26.1 limma_3.4.0 [5] Biobase_2.8.0 loaded via a namespace (and not attached): [1] affyio_1.16.0 > ###################################################################### ################### On Jim MacDonald's advice I tried a trace with the debug command: When I run the command: agiMicro.rma=rmaMicroRna(agiMicroraw,normalize=TRUE,background=FALSE) in debug mode, I see that after step: index = which(dd$genes$ProbeName %in% ProbeName.rep[ii]) when I print index it returns integer(0) which means that the ProbeName was not found. Consequently the step of median calculation returns NAs. In contrast when I run the same command with the example data dd.micro then the results after index = which(dd$genes$ProbeName %in% ProbeName.rep[ii]) and printing index returns values such as: 38 64 7162 7322 8113 8556 8883 9731 which is basically returning the ProbeName indices. Since for my data the index retuns a zero values is that the reason I am getting NA in the next steps and consequently at the step if (min(dd.aux$Rb) < 0) { dd.aux$Rb = dd.aux$Rb + abs(min(dd.aux$Rb)) + 0.5 which returns the error: Error in if (min(dd.aux$Rb) < 0) { : missing value where TRUE/FALSE needed It seems to me that the program is not reading in the probe name data correctly. I am using the mouse Agilent data. Has the program been tested on mouse as well as human? Also, a possible complicating factor is that the test example with the program does not include a step where the data is read in to form the files. The data is simply read in from the data statement, so I haven't really tried the program on reading in a test data set. My session up to the attempt to normalize is: ###################################################################### ########### > targets<-readTargets("Targets.txt",verbose=TRUE) Target File FileName Treatment GErep AxCNTx1x1x1 AxCNTx1x1x1.txt A.CNT 1 AxCNTx2x2x2 AxCNTx2x2x2.txt A.CNT 1 AxCNTx3x3x3 AxCNTx3x3x3.txt A.CNT 1 AxCNTx7x4x4 AxCNTx7x4x4.txt A.CNT 1 BxCCL4x1x1x5 BxCCL4x1x1x5.txt B.CCL4 2 BxCCL4x2x2x6 BxCCL4x2x2x6.txt B.CCL4 2 BxCCL4x3x3x7 BxCCL4x3x3x7.txt B.CCL4 2 BxCCL4x5x4x8 BxCCL4x5x4x8.txt B.CCL4 2 CxBDLx1x1x9 CxBDLx1x1x9.txt C.BDL 3 CxBDLx2x2x10 CxBDLx2x2x10.txt C.BDL 3 CxBDLx3x3x11 CxBDLx3x3x11.txt C.BDL 3 CxBDLx4x4x12 CxBDLx4x4x12.txt C.BDL 3 DxaHSCx1x1x13 DxaHSCx1x1x13.txt D.aHSC 4 DxaHSCx2x2x14 DxaHSCx2x2x14.txt D.aHSC 4 DxaHSCx3x3x15 DxaHSCx3x3x15.txt D.aHSC 4 DxaHSCx4x4x16 DxaHSCx4x4x16.txt D.aHSC 4 > agiMicroraw<-readMicroRnaAFE(targets,verbose=TRUE) Read AxCNTx1x1x1.txt Read AxCNTx2x2x2.txt Read AxCNTx3x3x3.txt Read AxCNTx7x4x4.txt Read BxCCL4x1x1x5.txt Read BxCCL4x2x2x6.txt Read BxCCL4x3x3x7.txt Read BxCCL4x5x4x8.txt Read CxBDLx1x1x9.txt Read CxBDLx2x2x10.txt Read CxBDLx3x3x11.txt Read CxBDLx4x4x12.txt Read DxaHSCx1x1x13.txt Read DxaHSCx2x2x14.txt Read DxaHSCx3x3x15.txt Read DxaHSCx4x4x16.txt RGList: dd$R: 'gTotalGeneSignal' dd$G: 'gTotalProbeSignal' dd$Rb: 'gMeanSignal' dd$Gb: 'gProcessedSignal' > agiMicro.rma=rmaMicroRna(agiMicroraw, + normalize=TRUE, + background=FALSE) Error in if (min(dd.aux$Rb) < 0) { : missing value where TRUE/FALSE needed #################################### I would greatly appreciate any help or suggestions you can offer. Best wishes, Rich ------------------------------------------------------------ Richard A. Friedman, PhD Associate Research Scientist, Biomedical Informatics Shared Resource Herbert Irving Comprehensive Cancer Center (HICCC) Lecturer, Department of Biomedical Informatics (DBMI) Educational Coordinator, Center for Computational Biology and Bioinformatics (C2B2)/ National Center for Multiscale Analysis of Genomic Networks (MAGNet) Room 824 Irving Cancer Research Center Columbia University 1130 St. Nicholas Ave New York, NY 10032 (212)851-4765 (voice) friedman at cancercenter.columbia.edu http://cancercenter.columbia.edu/~friedman/ In Memoriam, George Scithers

Dear Pedro and list, I believe that I may have found the problem, detailed below. I ran the GSE19232 test set used in Pedro's paper, which used the Agilent human microRNA array v 2.0 (G44708). The rmaMicroRna worked perfectly. My own array is the Mouse miRNA Microarray Release 12.0 G4471A-021828. It has 2 columns not in Agilent human microRNA array v 2.0 (G44708. I do not know if these are a matter of the the array, the software or the settings on the software. I will see if I can read in the data if I remove those columns by hand, with Excel but this will not be the best fix in the long run, if the program has a new ouput format. I will keep you posted. Best wishes, Rich On Jun 16, 2010, at 11:18 AM, Richard Friedman wrote: > Dear Pedro, Jim, and Bioconductor List, > > I received an error message when trying to normalize data with > AgiMicroRMA > > agiMicro.rma=rmaMicroRna(agiMicroraw, > + normalize=TRUE, > + background=FALSE) > Error in if (min(dd.aux$Rb) < 0) { : > missing value where TRUE/FALSE needed > > agiMicro.rma=rmaMicroRna(agiMicroraw, > + normalize=TRUE, > + background=TRUE) > Error in if (min(dd.aux$Rb) < 0) { : > missing value where TRUE/FALSE needed > > > #################################################################### ############ > My session Inforrmation is: > > > sessionInfo() > R version 2.11.0 (2010-04-22) > i386-apple-darwin9.8.0 > > locale: > [1] en_US.UTF-8/en_US.UTF-8/C/C/en_US.UTF-8/en_US.UTF-8 > > attached base packages: > [1] stats graphics grDevices utils datasets methods base > > other attached packages: > [1] AgiMicroRna_1.2.0 preprocessCore_1.10.0 > affy_1.26.1 limma_3.4.0 > [5] Biobase_2.8.0 > > loaded via a namespace (and not attached): > [1] affyio_1.16.0 > > > > #################################################################### ##################### > > On Jim MacDonald's advice I tried a trace with the debug command: > > When I run the command: > > agiMicro > .rma=rmaMicroRna(agiMicroraw,normalize=TRUE,background=FALSE) in > debug mode, I see that after step: > > index = which(dd$genes$ProbeName %in% ProbeName.rep[ii]) > when I print index it returns > integer(0) > which means that the ProbeName was not found. Consequently the step > of median calculation returns NAs. > > In contrast when I run the same command with the example data > dd.micro then the results after > index = which(dd$genes$ProbeName %in% ProbeName.rep[ii]) > and printing index returns values such as: > 38 64 7162 7322 8113 8556 8883 9731 > which is basically returning the ProbeName indices. > > Since for my data the index retuns a zero values is that the reason > I am getting NA in the next steps and consequently at the step > > if (min(dd.aux$Rb) < 0) { > dd.aux$Rb = dd.aux$Rb + abs(min(dd.aux$Rb)) + 0.5 > > which returns the error: > > Error in if (min(dd.aux$Rb) < 0) { : > missing value where TRUE/FALSE needed > > It seems to me that the program is not reading in the probe name > data correctly. > I am using the mouse Agilent data. > Has the program been tested on mouse as well as human? > Also, a possible complicating factor is that the test example with > the program does not include a step where > the data is read in to form the files. The data is simply read in > from the data statement, so I haven't really tried the > program on reading in a test data set. > > My session up to the attempt to normalize is: > #################################################################### ############# > > > > targets<-readTargets("Targets.txt",verbose=TRUE) > > Target File > FileName Treatment GErep > AxCNTx1x1x1 AxCNTx1x1x1.txt A.CNT 1 > AxCNTx2x2x2 AxCNTx2x2x2.txt A.CNT 1 > AxCNTx3x3x3 AxCNTx3x3x3.txt A.CNT 1 > AxCNTx7x4x4 AxCNTx7x4x4.txt A.CNT 1 > BxCCL4x1x1x5 BxCCL4x1x1x5.txt B.CCL4 2 > BxCCL4x2x2x6 BxCCL4x2x2x6.txt B.CCL4 2 > BxCCL4x3x3x7 BxCCL4x3x3x7.txt B.CCL4 2 > BxCCL4x5x4x8 BxCCL4x5x4x8.txt B.CCL4 2 > CxBDLx1x1x9 CxBDLx1x1x9.txt C.BDL 3 > CxBDLx2x2x10 CxBDLx2x2x10.txt C.BDL 3 > CxBDLx3x3x11 CxBDLx3x3x11.txt C.BDL 3 > CxBDLx4x4x12 CxBDLx4x4x12.txt C.BDL 3 > DxaHSCx1x1x13 DxaHSCx1x1x13.txt D.aHSC 4 > DxaHSCx2x2x14 DxaHSCx2x2x14.txt D.aHSC 4 > DxaHSCx3x3x15 DxaHSCx3x3x15.txt D.aHSC 4 > DxaHSCx4x4x16 DxaHSCx4x4x16.txt D.aHSC 4 > > > agiMicroraw<-readMicroRnaAFE(targets,verbose=TRUE) > Read AxCNTx1x1x1.txt > Read AxCNTx2x2x2.txt > Read AxCNTx3x3x3.txt > Read AxCNTx7x4x4.txt > Read BxCCL4x1x1x5.txt > Read BxCCL4x2x2x6.txt > Read BxCCL4x3x3x7.txt > Read BxCCL4x5x4x8.txt > Read CxBDLx1x1x9.txt > Read CxBDLx2x2x10.txt > Read CxBDLx3x3x11.txt > Read CxBDLx4x4x12.txt > Read DxaHSCx1x1x13.txt > Read DxaHSCx2x2x14.txt > Read DxaHSCx3x3x15.txt > Read DxaHSCx4x4x16.txt > > RGList: > dd$R: 'gTotalGeneSignal' > dd$G: 'gTotalProbeSignal' > dd$Rb: 'gMeanSignal' > dd$Gb: 'gProcessedSignal' > > > > > agiMicro.rma=rmaMicroRna(agiMicroraw, > + normalize=TRUE, > + background=FALSE) > Error in if (min(dd.aux$Rb) < 0) { : > missing value where TRUE/FALSE needed > #################################### > > I would greatly appreciate any help or suggestions you can offer. > > Best wishes, > Rich > ------------------------------------------------------------ > Richard A. Friedman, PhD > Associate Research Scientist, > Biomedical Informatics Shared Resource > Herbert Irving Comprehensive Cancer Center (HICCC) > Lecturer, > Department of Biomedical Informatics (DBMI) > Educational Coordinator, > Center for Computational Biology and Bioinformatics (C2B2)/ > National Center for Multiscale Analysis of Genomic Networks (MAGNet) > Room 824 > Irving Cancer Research Center > Columbia University > 1130 St. Nicholas Ave > New York, NY 10032 > (212)851-4765 (voice) > friedman at cancercenter.columbia.edu > http://cancercenter.columbia.edu/~friedman/ > > In Memoriam, > George Scithers > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor

Dear Richard, AgiMicroRna has not been tested on mouse data, something that I?m just doing right now. It seems that I am having problems myself loading the data into R. What version of Agilent Feature Extraction are you using?. The people that usually provide me with the scanned data files told me that they have updated the Agilent Feature Extraction from version 9.5.3.1 to 10.7.3.1, and this new version does not give columns such as "accessions", "chr_coord", "ProbeUID", "gBGUsed", "gMeanSignal". Can you confirm with me if this is true?, Honestly, I think that it is a bit weird that the new version of the Extractor doesn?t give the "gMeanSignal". For your problem, try this (assuming your RGList object is named "dd") to check if you can get the index vector containing the replicated probes. ProbeName.rep = unique(dd$genes$ProbeName[duplicated(dd$genes$ProbeName)]) length(ProbeName.rep ) for(ii in 1: length(ProbeName.rep)){ index=which(dd$genes$ProbeName %in% ProbeName.rep[ii]) cat(ii, " - ",index,"\n") } Let me know what u get from here. It might be helpful if you were able to send me a couple of your text files to do the checkings myself, and try to figure out where the problem is. p.- -----Mensaje original----- De: Richard Friedman [mailto:friedman at cancercenter.columbia.edu] Enviado el: Wednesday, June 16, 2010 5:18 PM Para: Pedro L?pez Romero; James W. MacDonald; bio c bioconductor Asunto: Error message with rmaMicroRna on mouse data in AgiMicroRNA Dear Pedro, Jim, and Bioconductor List, I received an error message when trying to normalize data with AgiMicroRMA > agiMicro.rma=rmaMicroRna(agiMicroraw, + normalize=TRUE, + background=FALSE) Error in if (min(dd.aux$Rb) < 0) { : missing value where TRUE/FALSE needed > agiMicro.rma=rmaMicroRna(agiMicroraw, + normalize=TRUE, + background=TRUE) Error in if (min(dd.aux$Rb) < 0) { : missing value where TRUE/FALSE needed > ###################################################################### ########## My session Inforrmation is: > sessionInfo() R version 2.11.0 (2010-04-22) i386-apple-darwin9.8.0 locale: [1] en_US.UTF-8/en_US.UTF-8/C/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] AgiMicroRna_1.2.0 preprocessCore_1.10.0 affy_1.26.1 limma_3.4.0 [5] Biobase_2.8.0 loaded via a namespace (and not attached): [1] affyio_1.16.0 > ###################################################################### ################### On Jim MacDonald's advice I tried a trace with the debug command: When I run the command: agiMicro.rma=rmaMicroRna(agiMicroraw,normalize=TRUE,background=FALSE) in debug mode, I see that after step: index = which(dd$genes$ProbeName %in% ProbeName.rep[ii]) when I print index it returns integer(0) which means that the ProbeName was not found. Consequently the step of median calculation returns NAs. In contrast when I run the same command with the example data dd.micro then the results after index = which(dd$genes$ProbeName %in% ProbeName.rep[ii]) and printing index returns values such as: 38 64 7162 7322 8113 8556 8883 9731 which is basically returning the ProbeName indices. Since for my data the index retuns a zero values is that the reason I am getting NA in the next steps and consequently at the step if (min(dd.aux$Rb) < 0) { dd.aux$Rb = dd.aux$Rb + abs(min(dd.aux$Rb)) + 0.5 which returns the error: Error in if (min(dd.aux$Rb) < 0) { : missing value where TRUE/FALSE needed It seems to me that the program is not reading in the probe name data correctly. I am using the mouse Agilent data. Has the program been tested on mouse as well as human? Also, a possible complicating factor is that the test example with the program does not include a step where the data is read in to form the files. The data is simply read in from the data statement, so I haven't really tried the program on reading in a test data set. My session up to the attempt to normalize is: ###################################################################### ########### > targets<-readTargets("Targets.txt",verbose=TRUE) Target File FileName Treatment GErep AxCNTx1x1x1 AxCNTx1x1x1.txt A.CNT 1 AxCNTx2x2x2 AxCNTx2x2x2.txt A.CNT 1 AxCNTx3x3x3 AxCNTx3x3x3.txt A.CNT 1 AxCNTx7x4x4 AxCNTx7x4x4.txt A.CNT 1 BxCCL4x1x1x5 BxCCL4x1x1x5.txt B.CCL4 2 BxCCL4x2x2x6 BxCCL4x2x2x6.txt B.CCL4 2 BxCCL4x3x3x7 BxCCL4x3x3x7.txt B.CCL4 2 BxCCL4x5x4x8 BxCCL4x5x4x8.txt B.CCL4 2 CxBDLx1x1x9 CxBDLx1x1x9.txt C.BDL 3 CxBDLx2x2x10 CxBDLx2x2x10.txt C.BDL 3 CxBDLx3x3x11 CxBDLx3x3x11.txt C.BDL 3 CxBDLx4x4x12 CxBDLx4x4x12.txt C.BDL 3 DxaHSCx1x1x13 DxaHSCx1x1x13.txt D.aHSC 4 DxaHSCx2x2x14 DxaHSCx2x2x14.txt D.aHSC 4 DxaHSCx3x3x15 DxaHSCx3x3x15.txt D.aHSC 4 DxaHSCx4x4x16 DxaHSCx4x4x16.txt D.aHSC 4 > agiMicroraw<-readMicroRnaAFE(targets,verbose=TRUE) Read AxCNTx1x1x1.txt Read AxCNTx2x2x2.txt Read AxCNTx3x3x3.txt Read AxCNTx7x4x4.txt Read BxCCL4x1x1x5.txt Read BxCCL4x2x2x6.txt Read BxCCL4x3x3x7.txt Read BxCCL4x5x4x8.txt Read CxBDLx1x1x9.txt Read CxBDLx2x2x10.txt Read CxBDLx3x3x11.txt Read CxBDLx4x4x12.txt Read DxaHSCx1x1x13.txt Read DxaHSCx2x2x14.txt Read DxaHSCx3x3x15.txt Read DxaHSCx4x4x16.txt RGList: dd$R: 'gTotalGeneSignal' dd$G: 'gTotalProbeSignal' dd$Rb: 'gMeanSignal' dd$Gb: 'gProcessedSignal' > agiMicro.rma=rmaMicroRna(agiMicroraw, + normalize=TRUE, + background=FALSE) Error in if (min(dd.aux$Rb) < 0) { : missing value where TRUE/FALSE needed #################################### I would greatly appreciate any help or suggestions you can offer. Best wishes, Rich ------------------------------------------------------------ Richard A. Friedman, PhD Associate Research Scientist, Biomedical Informatics Shared Resource Herbert Irving Comprehensive Cancer Center (HICCC) Lecturer, Department of Biomedical Informatics (DBMI) Educational Coordinator, Center for Computational Biology and Bioinformatics (C2B2)/ National Center for Multiscale Analysis of Genomic Networks (MAGNet) Room 824 Irving Cancer Research Center Columbia University 1130 St. Nicholas Ave New York, NY 10032 (212)851-4765 (voice) friedman at cancercenter.columbia.edu http://cancercenter.columbia.edu/~friedman/ In Memoriam, George Scithers *************** AVISO LEGAL *************** Este mensaje va dirigido, de manera exclusiva, a su destinatario y contiene informaci?n confidencial y sujeta al secreto profesional, cuya divulgaci?n no est? permitida por la ley. 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