I am interested in detecting splicing differences between 2 conditions, with 5 biological replicates of each, and DEXSeq seems like a good tool for the job. > ABsamples condition replicate A013 A 1 A014 A 2 A015 A 3 A016 A 4 A017 A 5 B013 B 1 B014 B 2 B015 B 3 B016 B 4 B017 B 5 I generated an exon count matrix for non-overlapping Ensemblv64 exon parts using the provided dexseq_prepare_annotation.py and BEDtools. (I used Tophat for alignment.) As far as I can tell, the ExonCountSet was created successfully from this input > ABMatrix <- read.table(file="ABExonsMatrix.txt") > ABgeneExonIDs <- read.table(file="ABExonIDs.txt") > ABsamples <- read.table("ABsamples.txt") > ABExon <- newExonCountSet(countData = ABMatrix, design = ABsamples, geneIDs = ABgeneExonIDs$V2, exonIDs = ABgeneExonIDs$V1) I proceeded to this step: > AB013to017 <- makeCompleteDEUAnalysis(ABExon, nCores=22) and the "Estimating Cox-Reid exon dispersion estimates" and the "Testing for differential exon usage" steps also looked like they worked (though there were a few errors like this for some of the exons "Error in chol.default(XVX + lambda * I) : the leading minor of order 10 is not positive definite", but I am pretty sure that isn't my underlying problem) But then this happened: Calculating fold changes using 22 cores. (Progress report: one dot per 100 genes) Error in function (classes, fdef, mtable) : unable to find an inherited method for function "fData", for signature "try-error" In addition: Warning message: In mcLapply(allecs, FUN = funtoapply, mc.cores = mc.cores) : all scheduled cores encountered errors in user code I am not a very experienced R user, so I am not sure if this error stems from problems with my dataset, problems with the underlying R environment, or a problem with DEXSeq itself. I would appreciate any thoughts and feedback you may have. Please let me know if I should provide additional information and I would be happy to do so. Thanks for your time, Beth -- output of sessionInfo(): > sessionInfo() R version 2.14.2 (2012-02-29) Platform: x86_64-unknown-linux-gnu (64-bit) locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 [7] LC_PAPER=C LC_NAME=C [9] LC_ADDRESS=C LC_TELEPHONE=C [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] multicore_0.1-7 DEXSeq_1.0.2 Biobase_2.14.0 [4] BiocInstaller_1.2.1 loaded via a namespace (and not attached): [1] hwriter_1.3 plyr_1.7.1 statmod_1.4.14 stringr_0.6 tools_2.14.2 -- Sent via the guest posting facility at bioconductor.org.

Hi Beth, Thanks for your email. This error comes from the glm function, sometimes it fails due to really high dispersions. e.g. cases where you have 0 counts in an exon in almost all your samples and one sample with 1000 counts. In my experience this rarely happens so it should not be a problem for the analysis. Could you try updating to the newest version of DEXSeq? This version should convert these error into warnings, if this does not work, could you send me your ExonCountSet object to give a closer look to the error message? Best wishes, Alejandro Reyes Date: Mon, 9 Apr 2012 10:05:52 -0700 (PDT) From: "Beth [guest]"<guest@bioconductor.org> To:bioconductor at r-project.org,elizzybethy at hotmail.com Subject: [BioC] DEXSeq error during fold change calculation Message-ID:<20120409170552.035491313D0 at mamba.fhcrc.org> I am interested in detecting splicing differences between 2 conditions, with 5 biological replicates of each, and DEXSeq seems like a good tool for the job. > ABsamples condition replicate A013 A 1 A014 A 2 A015 A 3 A016 A 4 A017 A 5 B013 B 1 B014 B 2 B015 B 3 B016 B 4 B017 B 5 I generated an exon count matrix for non-overlapping Ensemblv64 exon parts using the provided dexseq_prepare_annotation.py and BEDtools. (I used Tophat for alignment.) As far as I can tell, the ExonCountSet was created successfully from this input > ABMatrix<- read.table(file="ABExonsMatrix.txt") > ABgeneExonIDs<- read.table(file="ABExonIDs.txt") > ABsamples<- read.table("ABsamples.txt") > ABExon<- newExonCountSet(countData = ABMatrix, design = ABsamples, geneIDs = ABgeneExonIDs$V2, exonIDs = ABgeneExonIDs$V1) I proceeded to this step: > AB013to017<- makeCompleteDEUAnalysis(ABExon, nCores=22) and the "Estimating Cox-Reid exon dispersion estimates" and the "Testing for differential exon usage" steps also looked like they worked (though there were a few errors like this for some of the exons "Error in chol.default(XVX + lambda * I) : the leading minor of order 10 is not positive definite", but I am pretty sure that isn't my underlying problem) But then this happened: Calculating fold changes using 22 cores. (Progress report: one dot per 100 genes) Error in function (classes, fdef, mtable) : unable to find an inherited method for function "fData", for signature "try-error" In addition: Warning message: In mcLapply(allecs, FUN = funtoapply, mc.cores = mc.cores) : all scheduled cores encountered errors in user code I am not a very experienced R user, so I am not sure if this error stems from problems with my dataset, problems with the underlying R environment, or a problem with DEXSeq itself. I would appreciate any thoughts and feedback you may have. Please let me know if I should provide additional information and I would be happy to do so. Thanks for your time, Beth -- output of sessionInfo(): > sessionInfo() R version 2.14.2 (2012-02-29) Platform: x86_64-unknown-linux-gnu (64-bit) locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 [7] LC_PAPER=C LC_NAME=C [9] LC_ADDRESS=C LC_TELEPHONE=C [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] multicore_0.1-7 DEXSeq_1.0.2 Biobase_2.14.0 [4] BiocInstaller_1.2.1 loaded via a namespace (and not attached): [1] hwriter_1.3 plyr_1.7.1 statmod_1.4.14 stringr_0.6 tools_2.14.2

Dear Beth thank you for the feedback and detailed problem description! Can you try again with a more recent version of DEXSeq e.g. http://www.bioconductor.org/packages/release/bioc/html/DEXSeq.html (better even, the 'devel' version since the package is still continously being improved.) You have been using a relatively old and dusty version of DEXSeq, and I believe that there was a bug in the code that caused messages like the one you report (not leading to false results, but to fatal errors during the execution), and which has since long been fixed. Please let us know how it goes with the newer version. Best wishes Wolfgang Apr/9/12 7:05 PM, Beth [guest] scripsit:: > > I am interested in detecting splicing differences between 2 conditions, with 5 biological replicates of each, and DEXSeq seems like a good tool for the job. > >> ABsamples > condition replicate > A013 A 1 > A014 A 2 > A015 A 3 > A016 A 4 > A017 A 5 > B013 B 1 > B014 B 2 > B015 B 3 > B016 B 4 > B017 B 5 > > I generated an exon count matrix for non-overlapping Ensemblv64 exon parts using the provided dexseq_prepare_annotation.py and BEDtools. (I used Tophat for alignment.) > > As far as I can tell, the ExonCountSet was created successfully from this input >> ABMatrix<- read.table(file="ABExonsMatrix.txt") >> ABgeneExonIDs<- read.table(file="ABExonIDs.txt") >> ABsamples<- read.table("ABsamples.txt") >> ABExon<- newExonCountSet(countData = ABMatrix, design = ABsamples, geneIDs = ABgeneExonIDs$V2, exonIDs = ABgeneExonIDs$V1) > > I proceeded to this step: >> AB013to017<- makeCompleteDEUAnalysis(ABExon, nCores=22) > > and the "Estimating Cox-Reid exon dispersion estimates" and the "Testing for differential exon usage" steps also looked like they worked (though there were a few errors like this for some of the exons "Error in chol.default(XVX + lambda * I) : the leading minor of order 10 is not positive definite", but I am pretty sure that isn't my underlying problem) > > But then this happened: > Calculating fold changes using 22 cores. (Progress report: one dot per 100 genes) > Error in function (classes, fdef, mtable) : > unable to find an inherited method for function "fData", for signature "try-error" > In addition: Warning message: > In mcLapply(allecs, FUN = funtoapply, mc.cores = mc.cores) : > all scheduled cores encountered errors in user code > > I am not a very experienced R user, so I am not sure if this error stems from problems with my dataset, problems with the underlying R environment, or a problem with DEXSeq itself. > > I would appreciate any thoughts and feedback you may have. Please let me know if I should provide additional information and I would be happy to do so. > > Thanks for your time, > Beth > > -- output of sessionInfo(): > >> sessionInfo() > R version 2.14.2 (2012-02-29) > Platform: x86_64-unknown-linux-gnu (64-bit) > > locale: > [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C > [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 > [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 > [7] LC_PAPER=C LC_NAME=C > [9] LC_ADDRESS=C LC_TELEPHONE=C > [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C > > attached base packages: > [1] stats graphics grDevices utils datasets methods base > > other attached packages: > [1] multicore_0.1-7 DEXSeq_1.0.2 Biobase_2.14.0 > [4] BiocInstaller_1.2.1 > > loaded via a namespace (and not attached): > [1] hwriter_1.3 plyr_1.7.1 statmod_1.4.14 stringr_0.6 tools_2.14.2 > > > -- > 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 -- Best wishes Wolfgang Wolfgang Huber EMBL http://www.embl.de/research/units/genome_biology/huber