Dear All, I downloaded gene expression profiles of 132 laser microdissected colorectal cancer tissues from GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21815), from this dataset I would like to analyze the differential expression between the last 32 tumor and 9 normal tissues. I have no experience with this. I think the array is the single-color type. I loaded the data into R, and now I would like to run the function neqc() for background correction and normalization, however I am getting an error. Below I put my commands and the error statement. What am I doing wrong? Is the problem in making the EListRaw or the second step? What is alpha? I tried to find answers on the web but failed. Many thank! Femke library(limma) read.maimages(files=c("GSM543223.txt","GSM543224.txt","GSM543225.txt", "GSM543226.txt","GSM543227.txt","GSM543228.txt","GSM543229.txt","GSM54 3230.txt","GSM543231.txt","GSM543232.txt","GSM543233.txt","GSM543234.t xt","GSM543235.txt","GSM543236.txt","GSM543237.txt","GSM543238.txt","G SM543239.txt","GSM543240.txt","GSM543241.txt","GSM543242.txt","GSM5432 43.txt","GSM543244.txt","GSM543245.txt","GSM543246.txt","GSM543247.txt ","GSM543248.txt","GSM543249.txt","GSM543250.txt","GSM543251.txt","GSM 543252.txt","GSM543253.txt","GSM543254.txt","GSM543255.txt","GSM543256 .txt","GSM543257.txt","GSM543258.txt","GSM543259.txt","GSM543260.txt", "GSM543261.txt","GSM543262.txt","GSM543263.txt"), source="agilent", na mes=c("CRC_101","CRC_102","CRC_103","CRC_104","CRC_105","CRC_106","CRC _107","CRC_108","CRC_109","CRC_110","CRC_111","CRC_112","CRC_113","CRC _114","CRC_115","CRC_116","CRC_117","CRC_118","CRC_119","CRC_120","CRC _121","CRC_122","CRC_123","CRC_124","CRC_125","CRC_126","CRC_127","CRC _128","CRC _129","CRC_130","CRC_131","CRC_132","normal_1","normal_2","normal_3", "normal_4","normal_5","normal_6","normal_7","normal_8","normal_9"), channels=1) -> data neqc(data$E, status=data$genes$ControlType) Error in if (alpha <= 0) stop("alpha must be positive") : missing value where TRUE/FALSE needed -- output of sessionInfo(): R version 2.10.1 (2009-12-14) x86_64-pc-linux-gnu locale: [1] C attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] limma_3.2.1 loaded via a namespace (and not attached): [1] tools_2.10.1 -- Sent via the guest posting facility at bioconductor.org.

It's going to be tough to run neqc() without control probes (in fact I was under the impression that neqc was only for Illumina arrays, but I could be wrong). It's a neat dataset for sure, but if I look into it (I've been monkeying around with GEO and Agilent/Affy/Illumina/RNAseq conversions a lot lately), I get the feeling that you could have issues with processing it: > library(GEOquery) > Mori <- getGEO('GSE21815')[[1]] Found 1 file(s) GSE21815_series_matrix.txt.gz Using locally cached version: /tmp/RtmpZwVlZL/GSE21815_series_matrix.txt.gz Using locally cached version of GPL6480 found here: /tmp/RtmpZwVlZL/GPL6480.soft > grep('control', fvarLabels(Mori), ignore.case=TRUE, value=TRUE) [1] "CONTROL_TYPE" > table(fData(Mori)$CONTROL_TYPE) FALSE 41000 I could certainly be wrong, but this would seem to be a problem if you want to use neqc(). Hopefully Wei Shi or Gordon Smyth or someone who knows better will chime in... On Thu, Dec 8, 2011 at 5:14 AM, Femke [guest] <guest@bioconductor.org>wrote: > > Dear All, > > I downloaded gene expression profiles of 132 laser microdissected > colorectal cancer tissues from GEO ( > http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21815), from this > dataset I would like to analyze the differential expression between the > last 32 tumor and 9 normal tissues. I have no experience with this. I think > the array is the single-color type. > > I loaded the data into R, and now I would like to run the function neqc() > for background correction and normalization, however I am getting an error. > Below I put my commands and the error statement. What am I doing wrong? Is > the problem in making the EListRaw or the second step? What is alpha? I > tried to find answers on the web but failed. > > Many thank! > Femke > > library(limma) > > read.maimages(files=c("GSM543223.txt","GSM543224.txt","GSM543225.txt ","GSM543226.txt","GSM543227.txt","GSM543228.txt","GSM543229.txt","GSM 543230.txt","GSM543231.txt","GSM543232.txt","GSM543233.txt","GSM543234 .txt","GSM543235.txt","GSM543236.txt","GSM543237.txt","GSM543238.txt", "GSM543239.txt","GSM543240.txt","GSM543241.txt","GSM543242.txt","GSM54 3243.txt","GSM543244.txt","GSM543245.txt","GSM543246.txt","GSM543247.t xt","GSM543248.txt","GSM543249.txt","GSM543250.txt","GSM543251.txt","G SM543252.txt","GSM543253.txt","GSM543254.txt","GSM543255.txt","GSM5432 56.txt","GSM543257.txt","GSM543258.txt","GSM543259.txt","GSM543260.txt ","GSM543261.txt","GSM543262.txt","GSM543263.txt"), > source="agilent", > names=c("CRC_101","CRC_102","CRC_103","CRC_104","CRC_105","CRC_106", "CRC_107","CRC_108","CRC_109","CRC_110","CRC_111","CRC_112","CRC_113", "CRC_114","CRC_115","CRC_116","CRC_117","CRC_118","CRC_119","CRC_120", "CRC_121","CRC_122","CRC_123","CRC_124","CRC_125","CRC_126","CRC_127", "CRC_128","CRC > _129","CRC_130","CRC_131","CRC_132","normal_1","normal_2","normal_3 ","normal_4","normal_5","normal_6","normal_7","normal_8","normal_9"), > channels=1) -> data > > > neqc(data$E, status=data$genes$ControlType) > > Error in if (alpha <= 0) stop("alpha must be positive") : > missing value where TRUE/FALSE needed > > > -- output of sessionInfo(): > > R version 2.10.1 (2009-12-14) > x86_64-pc-linux-gnu > > locale: > [1] C > > attached base packages: > [1] stats graphics grDevices utils datasets methods base > > other attached packages: > [1] limma_3.2.1 > > loaded via a namespace (and not attached): > [1] tools_2.10.1 > > -- > Sent via the guest posting facility at bioconductor.org. > > _______________________________________________ > Bioconductor mailing list > Bioconductor@r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > -- If people do not believe that mathematics is simple, it is only because they do not realize how complicated life is. John von Neumann<http: www-groups.dcs.st-="" and.ac.uk="" ~history="" biographies="" von_neumann.html=""> [[alternative HTML version deleted]]

Dear Femke, Your R is 2 years old. Could you update your R and limma to the latest version and then rerun your code? We have successfully applied neqc function to the Agilent one-color array data which contains ~100 negative control probes (we used raw data). But I can't see any reason why it did not work on the GEO data. Cheers, Wei On Dec 9, 2011, at 12:14 AM, Femke [guest] wrote: > > Dear All, > > I downloaded gene expression profiles of 132 laser microdissected colorectal cancer tissues from GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21815), from this dataset I would like to analyze the differential expression between the last 32 tumor and 9 normal tissues. I have no experience with this. I think the array is the single-color type. > > I loaded the data into R, and now I would like to run the function neqc() for background correction and normalization, however I am getting an error. Below I put my commands and the error statement. What am I doing wrong? Is the problem in making the EListRaw or the second step? What is alpha? I tried to find answers on the web but failed. > > Many thank! > Femke > > library(limma) > > read.maimages(files=c("GSM543223.txt","GSM543224.txt","GSM543225.txt ","GSM543226.txt","GSM543227.txt","GSM543228.txt","GSM543229.txt","GSM 543230.txt","GSM543231.txt","GSM543232.txt","GSM543233.txt","GSM543234 .txt","GSM543235.txt","GSM543236.txt","GSM543237.txt","GSM543238.txt", "GSM543239.txt","GSM543240.txt","GSM543241.txt","GSM543242.txt","GSM54 3243.txt","GSM543244.txt","GSM543245.txt","GSM543246.txt","GSM543247.t xt","GSM543248.txt","GSM543249.txt","GSM543250.txt","GSM543251.txt","G SM543252.txt","GSM543253.txt","GSM543254.txt","GSM543255.txt","GSM5432 56.txt","GSM543257.txt","GSM543258.txt","GSM543259.txt","GSM543260.txt ","GSM543261.txt","GSM543262.txt","GSM543263.txt"), source="agilent", names=c("CRC_101","CRC_102","CRC_103","CRC_104","CRC_105","CRC_106","C RC_107","CRC_108","CRC_109","CRC_110","CRC_111","CRC_112","CRC_113","C RC_114","CRC_115","CRC_116","CRC_117","CRC_118","CRC_119","CRC_120","C RC_121","CRC_122","CRC_123","CRC_124","CRC_125","CRC_126","CRC_127","C RC_128","CRC > _129","CRC_130","CRC_131","CRC_132","normal_1","normal_2","normal_3" ,"normal_4","normal_5","normal_6","normal_7","normal_8","normal_9"), channels=1) -> data > > > neqc(data$E, status=data$genes$ControlType) > > Error in if (alpha <= 0) stop("alpha must be positive") : > missing value where TRUE/FALSE needed > > > -- output of sessionInfo(): > > R version 2.10.1 (2009-12-14) > x86_64-pc-linux-gnu > > locale: > [1] C > > attached base packages: > [1] stats graphics grDevices utils datasets methods base > > other attached packages: > [1] limma_3.2.1 > > loaded via a namespace (and not attached): > [1] tools_2.10.1 > > -- > Sent via the guest posting facility at bioconductor.org. > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:6}}