Dear all, I am currently trying to use easyRNASeq in order to create a DGElist object for edgeR. However, when I run the function on my samples (as shown below), I encounter an error message AFTER counting: Preparing output Calculating library sizes from column totals. Normalizing counts Error in .local(obj, ...) : You need to estimate the common dispersion before using this function. Here's my call the easyRNASeq function: dgeList <- easyRNASeq(organism="Hsapiens", annotationMethod="biomaRt", gapped=TRUE, count="exons", filesDirectory = "/media/sf_Storage1/RNAseq", pattern="*_mapping\\.bam$", recursive=T, normalize=TRUE, outputFormat="edgeR", conditions=conditions ) I am following the provided online manual which does not mention any dispersion estimation. Does anyone have an idea how to fix this? Best regards, Ren?? -- output of sessionInfo(): Output: ... Processing G_349_mapping.bam Updating the read length information. The alignments are gapped. Minimum length of 1 bp. Maximum length of 101 bp. Preparing output Calculating library sizes from column totals. Normalizing counts Error in .local(obj, ...) : You need to estimate the common dispersion before using this function. In addition: There were 33 warnings (use warnings() to see them) > sessionInfo() R version 2.15.2 (2012-10-26) Platform: x86_64-pc-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] parallel stats graphics grDevices utils datasets methods [8] base other attached packages: [1] BSgenome.Hsapiens.UCSC.hg19_1.3.19 easyRNASeq_1.4.2 [3] ShortRead_1.16.1 latticeExtra_0.6-24 [5] RColorBrewer_1.0-5 Rsamtools_1.10.1 [7] DESeq_1.10.1 lattice_0.20-10 [9] locfit_1.5-8 BSgenome_1.26.1 [11] GenomicRanges_1.10.3 Biostrings_2.26.2 [13] IRanges_1.16.3 edgeR_3.0.2 [15] limma_3.14.1 biomaRt_2.14.0 [17] Biobase_2.18.0 genomeIntervals_1.14.0 [19] BiocGenerics_0.4.0 intervals_0.13.3 loaded via a namespace (and not attached): [1] annotate_1.36.0 AnnotationDbi_1.20.2 bitops_1.0-4.1 [4] DBI_0.2-5 genefilter_1.40.0 geneplotter_1.36.0 [7] grid_2.15.2 hwriter_1.3 RCurl_1.95-1.1 [10] RSQLite_0.11.2 splines_2.15.2 stats4_2.15.2 [13] survival_2.36-14 tools_2.15.2 XML_3.95-0.1 [16] xtable_1.7-0 zlibbioc_1.4.0 -- Sent via the guest posting facility at bioconductor.org.

Hi Ren?, Can you try to run it with normalize=FALSE? That should get you back a DGEList. The normalize=TRUE is a flag that start the edgeR processing (estimating the common dispersion, etc.) and performs some of the QA as described in the edgeR manual. You'll have to do these steps by yourself then. In the meanwhile (thanks for the report!), I'll check what broke down. I'll contact you again if I need more information from you to reproduce the error. Best, Nico --------------------------------------------------------------- Nicolas Delhomme Genome Biology Computational Support European Molecular Biology Laboratory Tel: +49 6221 387 8310 Email: nicolas.delhomme at embl.de Meyerhofstrasse 1 - Postfach 10.2209 69102 Heidelberg, Germany --------------------------------------------------------------- On Nov 19, 2012, at 9:29 AM, Ren? B?ttcher [guest] wrote: > > Dear all, > > I am currently trying to use easyRNASeq in order to create a DGElist object for edgeR. However, when I run the function on my samples (as shown below), I encounter an error message AFTER counting: > > Preparing output > Calculating library sizes from column totals. > Normalizing counts > Error in .local(obj, ...) : > You need to estimate the common dispersion before using this function. > > Here's my call the easyRNASeq function: > dgeList <- easyRNASeq(organism="Hsapiens", > annotationMethod="biomaRt", > gapped=TRUE, > count="exons", > filesDirectory = "/media/sf_Storage1/RNAseq", > pattern="*_mapping\\.bam$", > recursive=T, > normalize=TRUE, > outputFormat="edgeR", > conditions=conditions > ) > > I am following the provided online manual which does not mention any dispersion estimation. Does anyone have an idea how to fix this? > > Best regards, > Ren? > > -- output of sessionInfo(): > > Output: > ... > Processing G_349_mapping.bam > Updating the read length information. > The alignments are gapped. > Minimum length of 1 bp. > Maximum length of 101 bp. > Preparing output > Calculating library sizes from column totals. > Normalizing counts > Error in .local(obj, ...) : > You need to estimate the common dispersion before using this function. > In addition: There were 33 warnings (use warnings() to see them) > > >> sessionInfo() > R version 2.15.2 (2012-10-26) > Platform: x86_64-pc-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] parallel stats graphics grDevices utils datasets methods > [8] base > > other attached packages: > [1] BSgenome.Hsapiens.UCSC.hg19_1.3.19 easyRNASeq_1.4.2 > [3] ShortRead_1.16.1 latticeExtra_0.6-24 > [5] RColorBrewer_1.0-5 Rsamtools_1.10.1 > [7] DESeq_1.10.1 lattice_0.20-10 > [9] locfit_1.5-8 BSgenome_1.26.1 > [11] GenomicRanges_1.10.3 Biostrings_2.26.2 > [13] IRanges_1.16.3 edgeR_3.0.2 > [15] limma_3.14.1 biomaRt_2.14.0 > [17] Biobase_2.18.0 genomeIntervals_1.14.0 > [19] BiocGenerics_0.4.0 intervals_0.13.3 > > loaded via a namespace (and not attached): > [1] annotate_1.36.0 AnnotationDbi_1.20.2 bitops_1.0-4.1 > [4] DBI_0.2-5 genefilter_1.40.0 geneplotter_1.36.0 > [7] grid_2.15.2 hwriter_1.3 RCurl_1.95-1.1 > [10] RSQLite_0.11.2 splines_2.15.2 stats4_2.15.2 > [13] survival_2.36-14 tools_2.15.2 XML_3.95-0.1 > [16] xtable_1.7-0 zlibbioc_1.4.0 > > > -- > Sent via the guest posting facility at bioconductor.org.