Anne-Marie Madore <anne-marie.mador...@ulaval.ca> writes: > Hi, > > > > I am a Ph.D. student from Québec, Canada. Im a beginner with R and > Bioconductor. Until now the only experience I have is in analyzing
Please ask Bioconductor questions on the Bioconductor mailing list. http://bioconductor.org/docs/mailList.html Martin > microarray data using affy and limma packages. Now I am trying to analyze > Rat Gene 10 st arrays and I would like to run RMA analysis and Smyth > moderated t test on those arrays. Since no cdf official package is available > for those arrays, after reading many of the questions and responses on this > mailing list, I decided to use pdInfoBuilder, oligo and limma packages to > run analysis. The problem is, at the end, I get expression and differential > expression measured for all probe separately but not the calculated > expression representing all probe of each gene. When I run RMA, I got only > two steps, Background correcting and Normalizing but not Calculating > expression. Do you know how I can get differential expression calculated for > each gene? I dont know if the problem is in the package I built or if I can > use some code to answer this question. I list all codes used to build and > install the package pd.ragene.1.0.st.v1 and used to analyze expression > arrays below. > > > > Many thanks for your help, > > > > Anne-Marie Madore > > > > > > > > > > ## building the package > > > >> library(Biobase) > > Loading required package: tools > > > > Welcome to Bioconductor > > > > Vignettes contain introductory material. To view, type > > 'openVignette()'. To cite Bioconductor, see > > 'citation("Biobase")' and for packages 'citation(pkgname)'. > > > >> library(pdInfoBuilder) > > Loading required package: RSQLite > > Loading required package: DBI > > Loading required package: affxparser > > Loading required package: oligo > > Loading required package: splines > > Loading required package: preprocessCore > > Loading required package: AnnotationDbi > > Loading required package: oligoClasses > > oligo Package - Series 1.5.x > >> setwd("D:/Anne-Marie/Doctorat/puces ADN macrophages/puces rat/Annie > Dube/Analyse") > >> transFile <- > "RaGene-1_0-st-v1.na27.rn4.transcript.csv1/RaGene-1_0-st-v1.na27.rn4.transcr > ipt.csv" > >> probeFile <- "RaGene-1_0-st-v1.probe.tab/RaGene-1_0-st-v1.probe.tab" > >> clfFile <- "RaGene-1_0-st-v1.r4.clf/RaGene-1_0-st-v1.r4.clf" > >> pgfFile <- "RaGene-1_0-st-v1.r4.pgf/RaGene-1_0-st-v1.r4.pgf" > >> pkg <- new("AffyGenePDInfoPkgSeed", author="Anne-Marie Madore", > email="anne-marie.mador...@ulaval.ca", version="0.0.1", > > + genomebuild="RefSeq April 3, 2007, GenBank® January 25, 2007, Rat Ensembl > transcripts April 3, 2007 ", > > + biocViews="AnnotationData", pgfFile=pgfFile, clfFile=clfFile, > transFile=transFile, probeFile=probeFile) > >> makePdInfoPackage(pkg, destDir=".") > > Creating package in ./pd.ragene.1.0.st.v1 > > loadUnitsByBatch took 50.51 sec > > loadAffyCsv took 12.73 sec > > loadAffySeqCsv took 57.62 sec > > DB sort, index creation took 24.75 sec > > [1] TRUE > > Warning messages: > > 1: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL' > > 2: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL' > > > > > > ## installing the package in cmd command shell > > > > Microsoft Windows [version 6.0.6001] > > Copyright (c) 2006 Microsoft Corporation. Tous droits réservés. > > > > C:\Users\Anne-Marie Madore>cd c:\Program Files\R\R-2.8.1\bin > > > > c:\Program Files\R\R-2.8.1\bin>R CMD INSTALL pd.ragene.1.0.st.v1 > > installing to 'c:/PROGRA~1/R/R-28~1.1/library' > > > > > > ---------- Making package pd.ragene.1.0.st.v1 ------------ > > adding build stamp to DESCRIPTION > > installing NAMESPACE file and metadata > > installing R files > > installing inst files > > preparing package pd.ragene.1.0.st.v1 for lazy loading > > Loading required package: RSQLite > > Loading required package: DBI > > Loading required package: oligoClasses > > Loading required package: Biobase > > Loading required package: tools > > > > Welcome to Bioconductor > > > > Vignettes contain introductory material. To view, type > > 'openVignette()'. To cite Bioconductor, see > > 'citation("Biobase")' and for packages 'citation(pkgname)'. > > > > no man files in this package > > installing indices > > installing help > > adding MD5 sums > > > > * DONE (pd.ragene.1.0.st.v1) > > > > > > ## If I run a check (R CMD check pd.ragene.st.v1) I get three warning > messages and one note: > > > > 1. * checking R files for non-ASCII characters ... WARNING > Found the following files with non-ASCII characters: all.R Portable packages > must use only ASCII characters in their R code, except perhaps in comments. > > 2. * checking whether the name space can be loaded with stated > dependencies ... WARNING > Error in initDbConnection() : could not find function "dbConnect" Error: > .onLoad failed in 'loadNamespace' for 'pd.ragene.1.0.st.v1' Execution halted > A namespace must be able to be loaded with just the base namespace loaded: > otherwise if the namespace gets loaded by a saved object, the session will > be unable to start. > Probably some imports need to be declared in the NAMESPACE file. > > 3. * checking R code for possible problems ... NOTE > closeDb: no visible binding for global variable 'dbCon' > > 4. * checking for missing documentation entries ... WARNING > Undocumented code objects: > pd.ragene.1.0.st.v1 > All user-level objects in a package should have documentation entries. See > the chapter 'Writing R documentation files' in manual 'Writing R > Extensions'. > > > > > > ## analyzing the package > > > >> library("pd.ragene.1.0.st.v1") > >> library(oligo) > >> library(limma) > >> library(genefilter) > > Loading required package: survival > >> library(geneplotter) > > Loading required package: lattice > > Loading required package: annotate > > Loading required package: xtable > > KernSmooth 2.22 installed > > Copyright M. P. Wand 1997 > >> cel.files <- list.celfiles(".", full.names = TRUE) > >> basename(cel.files) > > [1] "AD_Ctrl_1.CEL" "AD_Ctrl_2.CEL" "AD_Ctrl_3.CEL" > "AD_Ctrl_5.CEL" > > [5] "AD_Ctrl_6.CEL" "AD_Traite_10.CEL" "AD_Traite_11.CEL" > "AD_Traite_7.CEL" > > [9] "AD_Traite_8.CEL" "AD_Traite_9.CEL" > >> test <- read.celfiles(cel.files) > > Platform design info loaded. > >> phenoData(test) <- read.AnnotatedDataFrame("phenoData.txt", header = TRUE, > row.name=1) > >> class(test) > > [1] "GeneFeatureSet" > > attr(,"package") > > [1] "oligoClasses" > >> class(phenoData) > > [1] "standardGeneric" > > attr(,"package") > > [1] "methods" > >> eset <- rma(test) > > Background correcting > > Normalizing > >> e <- exprs(eset) > >> index1 <- 1:5 > >> index2 <- 6:10 > >> d <- rowMeans(e[, index1]) - rowMeans(e[, index2]) > >> design <- model.matrix(~factor(eset$Key)) > >> fit <- lmFit(eset, design) > >> ebayes <- eBayes(fit) > >> sample <- row.names(ebayes) > >> Pvalue <- ebayes$p.value[,2] > >> Mean1 <- rowMeans(e[,index1]) > >> Mean2 <- rowMeans(e[,index2]) > >> sd1 <- apply(e[,index1], 1, "sd") > >> sd2 <- apply(e[,index2], 1, "sd") > >> csv <- read.csv(file="D:/Anne-Marie/Doctorat/puces ADN macrophages/puces > rat/Annie > Dube/Analyse/RaGene-1_0-st-v1.na27.rn4.transcript.header.DOS.csv",head=TRUE, > sep=",") > >> all <- merge(csv, Pvalue, by.x="sample", by.y=0, all=T) > >> all2 <- merge(all, Mean1, by.x="sample", by.y=0, all=T) > >> all3 <- merge(all2, Mean2, by.x="sample", by.y=0, all=T) > >> all4 <- merge(all3, sd1, by.x="sample", by.y=0, all=T) > >> all5 <- merge(all4, sd2, by.x="sample", by.y=0, all=T) > >> all6 <- merge(all5, d, by.x="sample", by.y=0, all=T) > >> write.table(data.frame(all6), file="ULTIMETESTtout.xls", sep="\t", > col.names = NA) > >> dim(all6) > > [1] 240410 24 > >> > >> # I also try using slightly the same way as I used with affy package > >> > >> pd <- read.AnnotatedDataFrame("pheno.txt", header = TRUE, row.names = 1) > >> pData(pd) > > Phenotype > > AD_Ctrl_1.CEL Control > > AD_Ctrl_2.CEL Control > > AD_Ctrl_3.CEL Control > > AD_Ctrl_5.CEL Control > > AD_Ctrl_6.CEL Control > > AD_Traite_7.CEL Traited > > AD_Traite_8.CEL Traited > > AD_Traite_9.CEL Traited > > AD_Traite_10.CEL Traited > > AD_Traite_11.CEL Traited > >> a <- read.celfiles(filenames = rownames(pData(pd)), phenoData = pd, > verbose = TRUE) > > Platform design info loaded. > >> eset <- rma(a) > > Background correcting > > Normalizing > >> exprs.eset <- exprs(eset) > >> index1 <- 1:5 > >> index2 <- 6:10 > >> d <- (rowMeans(exprs.eset[,index1]) - rowMeans(exprs.eset[,index2])) > >> population.groups <- factor (c(rep("Control",5), rep ("Traited",5))) > >> design <- model.matrix (~population.groups) > >> fit <- lmFit (eset, design) > >> fit.eBayes <- eBayes (fit) > >> sample <- row.names(fit.eBayes) > >> Pvalue <- fit.eBayes$p.value[,2] > >> Mean1 <- rowMeans(e[,index1]) > >> Mean2 <- rowMeans(e[,index2]) > >> sd1 <- apply(e[,index1], 1, "sd") > >> sd2 <- apply(e[,index2], 1, "sd") > >> csv <- read.csv(file="D:/Anne-Marie/Doctorat/puces ADN macrophages/puces > rat/Annie > Dube/Analyse/RaGene-1_0-st-v1.na27.rn4.transcript.header.DOS.csv",head=TRUE, > sep=",") > >> all <- merge(csv, Pvalue, by.x="sample", by.y=0, all=T) > >> all2 <- merge(all, Mean1, by.x="sample", by.y=0, all=T) > >> all3 <- merge(all2, Mean2, by.x="sample", by.y=0, all=T) > >> all4 <- merge(all3, sd1, by.x="sample", by.y=0, all=T) > >> all5 <- merge(all4, sd2, by.x="sample", by.y=0, all=T) > >> all6 <- merge(all5, d, by.x="sample", by.y=0, all=T) > >> write.table(data.frame(all6), file="TESTtout.xls", sep="\t", col.names = > NA) > >> dim(all6) > > [1] 240410 24 > >> sessionInfo() > > R version 2.8.1 (2008-12-22) > > i386-pc-mingw32 > > > > locale: > > LC_COLLATE=English_United States.1252;LC_CTYPE=English_United > States.1252;LC_MONETARY=English_United > States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252 > > > > attached base packages: > > [1] splines tools stats graphics grDevices utils datasets > methods base > > > > other attached packages: > > [1] geneplotter_1.20.0 annotate_1.20.1 xtable_1.5-4 > > > [4] lattice_0.17-17 genefilter_1.22.0 survival_2.34-1 > > > [7] limma_2.16.3 pd.ragene.1.0.st.v1_0.0.1 pdInfoBuilder_1.6.0 > > > [10] oligo_1.6.0 oligoClasses_1.4.0 AnnotationDbi_1.4.2 > > > [13] preprocessCore_1.4.0 affxparser_1.14.2 RSQLite_0.7-1 > > > [16] DBI_0.2-4 Biobase_2.2.1 > > > > loaded via a namespace (and not attached): > > [1] grid_2.8.1 KernSmooth_2.22-22 RColorBrewer_1.0-2 > > > > > [[alternative HTML version deleted]] > > ______________________________________________ > R-help@r-project.org mailing list > https://stat.ethz.ch/mailman/listinfo/r-help > PLEASE do read the posting guide http://www.R-project.org/posting-guide.html > and provide commented, minimal, self-contained, reproducible code. -- Martin Morgan Computational Biology / Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N. PO Box 19024 Seattle, WA 98109 Location: Arnold Building M2 B169 Phone: (206) 667-2793 ______________________________________________ R-help@r-project.org mailing list https://stat.ethz.ch/mailman/listinfo/r-help PLEASE do read the posting guide http://www.R-project.org/posting-guide.html and provide commented, minimal, self-contained, reproducible code.
