Hi Nico, I would like to use easyRNAseq to count reads for RNA-seq. The vignette says that "The ideal solution is to provide an annotation object that contains no overlapping features. The disjoin function from the IRanges package off ers a way to achieve this." I do not have much experience with IRanges, etc, and would be grateful for any pointers on how to do this, since it was not obvious to me. I would like to not remove the genes that overlap, but instead remove the regions of the genes that overlap, leaving any unique regions. And one additional request--would it be possible to have easyRNAseq have the option to calculate TPM as well as RPKM (using the non-overlapping gene length) ? The reference for TPM is http://www.ncbi.nlm.nih.gov/pubmed/22872506 TPM/RPKM would be useful for heatmaps and other display purposes. Thanks, Vince library(easyRNASeq) library(RnaSeqTutorial) library(BSgenome.Dmelanogaster.UCSC.dm3) count.table <- easyRNASeq(system.file( "extdata", package="RnaSeqTutorial"), organism="Dmelanogaster", readLength=36L, annotationMethod="gff", annotationFile=system.file( "extdata", "annot.gff", package="RnaSeqTutorial"), gapped=TRUE, count="exons", filenames="gapped.bam") > 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.Dmelanogaster.UCSC.dm3_1.3.19 [2] RnaSeqTutorial_0.0.11 [3] easyRNASeq_1.4.2 [4] ShortRead_1.16.3 [5] latticeExtra_0.6-24 [6] RColorBrewer_1.0-5 [7] Rsamtools_1.10.2 [8] DESeq_1.10.1 [9] lattice_0.20-13 [10] locfit_1.5-8 [11] BSgenome_1.26.1 [12] GenomicRanges_1.10.5 [13] Biostrings_2.26.2 [14] IRanges_1.16.4 [15] edgeR_3.0.8 [16] limma_3.14.3 [17] biomaRt_2.14.0 [18] Biobase_2.18.0 [19] genomeIntervals_1.14.0 [20] BiocGenerics_0.4.0 [21] intervals_0.13.3 [22] BiocInstaller_1.8.3 loaded via a namespace (and not attached): [1] annotate_1.36.0 AnnotationDbi_1.20.3 bitops_1.0-5 [4] DBI_0.2-5 genefilter_1.40.0 geneplotter_1.36.0 [7] grid_2.15.2 hwriter_1.3 RCurl_1.95-3 [10] RSQLite_0.11.2 splines_2.15.2 stats4_2.15.2 [13] survival_2.37-2 tools_2.15.2 XML_3.95-0.1 [16] xtable_1.7-0 zlibbioc_1.4.0

Hi Vincent, I'm currently doing a lot of refactoring on easyRNASeq and I should come with an example on how to achieve that soon in the developer version (1.5.x). I might propagate it to the stable version as a new function (1.4.x) as well, provided that it does not affect the stability in any way. I'm planning to add FPKM and I can obviously add TPM. These modification would probably only occur in the developer version (1.5.x) though. I can let you know once these are done, as well as how to get the developer version of Bioconductor if you're not familiar with that. Cheers, 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 Jan 9, 2013, at 3:23 PM, Vincent Schulz wrote: > Hi Nico, > > I would like to use easyRNAseq to count reads for RNA-seq. The vignette says that "The ideal solution is to provide an annotation object that contains no overlapping features. The disjoin function from the IRanges package off ers a way to achieve this." I do not have much experience with IRanges, etc, and would be grateful for any pointers on how to do this, since it was not obvious to me. I would like to not remove the genes that overlap, but instead remove the regions of the genes that overlap, leaving any unique regions. And one additional request-- would it be possible to have easyRNAseq have the option to calculate TPM as well as RPKM (using the non-overlapping gene length) ? The reference for TPM is > http://www.ncbi.nlm.nih.gov/pubmed/22872506 > TPM/RPKM would be useful for heatmaps and other display purposes. > > Thanks, > > Vince > > > library(easyRNASeq) > library(RnaSeqTutorial) > library(BSgenome.Dmelanogaster.UCSC.dm3) > > count.table <- easyRNASeq(system.file( > "extdata", > package="RnaSeqTutorial"), > organism="Dmelanogaster", > readLength=36L, > annotationMethod="gff", > annotationFile=system.file( > "extdata", > "annot.gff", > package="RnaSeqTutorial"), > gapped=TRUE, > count="exons", > filenames="gapped.bam") > > > 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.Dmelanogaster.UCSC.dm3_1.3.19 > [2] RnaSeqTutorial_0.0.11 > [3] easyRNASeq_1.4.2 > [4] ShortRead_1.16.3 > [5] latticeExtra_0.6-24 > [6] RColorBrewer_1.0-5 > [7] Rsamtools_1.10.2 > [8] DESeq_1.10.1 > [9] lattice_0.20-13 > [10] locfit_1.5-8 > [11] BSgenome_1.26.1 > [12] GenomicRanges_1.10.5 > [13] Biostrings_2.26.2 > [14] IRanges_1.16.4 > [15] edgeR_3.0.8 > [16] limma_3.14.3 > [17] biomaRt_2.14.0 > [18] Biobase_2.18.0 > [19] genomeIntervals_1.14.0 > [20] BiocGenerics_0.4.0 > [21] intervals_0.13.3 > [22] BiocInstaller_1.8.3 > > loaded via a namespace (and not attached): > [1] annotate_1.36.0 AnnotationDbi_1.20.3 bitops_1.0-5 > [4] DBI_0.2-5 genefilter_1.40.0 geneplotter_1.36.0 > [7] grid_2.15.2 hwriter_1.3 RCurl_1.95-3 > [10] RSQLite_0.11.2 splines_2.15.2 stats4_2.15.2 > [13] survival_2.37-2 tools_2.15.2 XML_3.95-0.1 > [16] xtable_1.7-0 zlibbioc_1.4.0