Dear Nico, Sorry to disturb you again. I am trying to use easyRNASeq to calculate fpkm values. I used the sample from the vignette, but all I get are "NA". Below are the details: > count.table <- easyRNASeq(system.file( + "extdata", + package="RnaSeqTutorial"), + organism="Dmelanogaster", + readLength=30L, + annotationMethod="rda", + annotationFile=system.file( + "data", + "gAnnot.rda", + package="RnaSeqTutorial"), + count="exons", + filenames=c("ACACTG.bam", "ACTAGC.bam", + "ATGGCT.bam", "TTGCGA.bam"), + normalize=TRUE + ) Checking arguments... Fetching annotations... Summarizing counts... Processing ACACTG.bam Processing ACTAGC.bam Processing ATGGCT.bam Processing TTGCGA.bam Preparing output Normalizing counts Warning messages: 1: In easyRNASeq(system.file("extdata", package = "RnaSeqTutorial"), : There are 50573 features/exons defined in your annotation that overlap! This implies that some reads will be counted more than once! Is that really what you want? 2: In easyRNASeq(system.file("extdata", package = "RnaSeqTutorial"), : You enforce UCSC chromosome conventions, however the provided annotation is not compliant. Correcting it. 3: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. 4: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. 5: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. 6: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. > head(count.table) ACACTG.bam ACTAGC.bam ATGGCT.bam TTGCGA.bam CG11023:1 NA NA NA NA CG11023:2 NA NA NA NA CG11023:3 NA NA NA NA CG2671:1 NA NA NA NA CG2671:2 NA NA NA NA CG2671:3 NA NA NA NA while the easyRNASeq works well without normalize=TRUE: > head(count.table) ACACTG.bam ACTAGC.bam ATGGCT.bam TTGCGA.bam CG11023:1 0 0 0 0 CG11023:2 0 0 0 0 CG11023:3 0 0 0 1 CG2671:1 0 0 0 0 CG2671:2 1 0 0 1 CG2671:3 13 8 11 12 I do not know what is wrong with my program. When I am trying to use my data, I also only get "NA" in the result. -- output of sessionInfo(): > sessionInfo() R version 2.15.1 (2012-06-22) Platform: i386-pc-mingw32/i386 (32-bit) locale: [1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252 [4] LC_NUMERIC=C LC_TIME=English_United States.1252 attached base packages: [1] parallel stats graphics grDevices utils datasets methods base other attached packages: [1] BSgenome.Dmelanogaster.UCSC.dm3_1.3.19 RnaSeqTutorial_0.0.11 easyRNASeq_1.4.2 [4] ShortRead_1.16.4 latticeExtra_0.6-24 RColorBrewer_1.0-5 [7] Rsamtools_1.10.2 DESeq_1.10.1 lattice_0.20-14 [10] locfit_1.5-8 BSgenome_1.26.1 GenomicRanges_1.10.7 [13] Biostrings_2.26.3 IRanges_1.16.6 edgeR_3.0.8 [16] limma_3.14.4 biomaRt_2.14.0 Biobase_2.18.0 [19] genomeIntervals_1.14.0 BiocGenerics_0.4.0 intervals_0.14.0 loaded via a namespace (and not attached): [1] annotate_1.36.0 AnnotationDbi_1.20.7 bitops_1.0-5 DBI_0.2-5 genefilter_1.40.0 geneplotter_1.36.0 grid_2.15.1 [8] hwriter_1.3 RCurl_1.95-4.1 RSQLite_0.11.2 splines_2.15.1 stats4_2.15.1 survival_2.37-4 tools_2.15.1 [15] XML_3.95-0.2 xtable_1.7-1 zlibbioc_1.4.0 -- Sent via the guest posting facility at bioconductor.org.

Dear Fuyan, No worries. You seem to have put the finger on a bug, I'll check that out. A new version of R and Bioc is available and any change I would do would go into that new version (will be 1.6.1 then). As we discussed offline, remember that easyRNASeq won't give you FPKM sensus-stricto. What you really get are RPKMs. Nico --------------------------------------------------------------- Nicolas Delhomme Nathaniel Street Lab Department of Plant Physiology Ume? Plant Science Center Tel: +46 90 786 7989 Email: nicolas.delhomme at plantphys.umu.se SLU - Ume? universitet Ume? S-901 87 Sweden --------------------------------------------------------------- On Apr 9, 2013, at 11:13 AM, Fuyan [guest] wrote: > > Dear Nico, > > Sorry to disturb you again. > > I am trying to use easyRNASeq to calculate fpkm values. > > > > I used the sample from the vignette, but all I get are "NA". > > Below are the details: > > > > >> count.table <- easyRNASeq(system.file( > + "extdata", > + package="RnaSeqTutorial"), > + organism="Dmelanogaster", > + readLength=30L, > + annotationMethod="rda", > + annotationFile=system.file( > + "data", > + "gAnnot.rda", > + package="RnaSeqTutorial"), > + count="exons", > + filenames=c("ACACTG.bam", "ACTAGC.bam", > + "ATGGCT.bam", "TTGCGA.bam"), > + normalize=TRUE > + ) > Checking arguments... > Fetching annotations... > Summarizing counts... > Processing ACACTG.bam > Processing ACTAGC.bam > Processing ATGGCT.bam > Processing TTGCGA.bam > Preparing output > Normalizing counts > Warning messages: > 1: In easyRNASeq(system.file("extdata", package = "RnaSeqTutorial"), : > There are 50573 features/exons defined in your annotation that overlap! This implies that some reads will be counted more than once! Is that really what you want? > 2: In easyRNASeq(system.file("extdata", package = "RnaSeqTutorial"), : > You enforce UCSC chromosome conventions, however the provided annotation is not compliant. Correcting it. > 3: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : > You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. > 4: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : > You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. > 5: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : > You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. > 6: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : > You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. > > > > >> head(count.table) > ACACTG.bam ACTAGC.bam ATGGCT.bam TTGCGA.bam > CG11023:1 NA NA NA NA > CG11023:2 NA NA NA NA > CG11023:3 NA NA NA NA > CG2671:1 NA NA NA NA > CG2671:2 NA NA NA NA > CG2671:3 NA NA NA NA > > > > while the easyRNASeq works well without normalize=TRUE: >> head(count.table) > ACACTG.bam ACTAGC.bam ATGGCT.bam TTGCGA.bam > CG11023:1 0 0 0 0 > CG11023:2 0 0 0 0 > CG11023:3 0 0 0 1 > CG2671:1 0 0 0 0 > CG2671:2 1 0 0 1 > CG2671:3 13 8 11 12 > > > > > > > I do not know what is wrong with my program. > > When I am trying to use my data, I also only get "NA" in the result. > > -- output of sessionInfo(): > >> sessionInfo() > R version 2.15.1 (2012-06-22) > Platform: i386-pc-mingw32/i386 (32-bit) > > locale: > [1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252 > [4] LC_NUMERIC=C LC_TIME=English_United States.1252 > > attached base packages: > [1] parallel stats graphics grDevices utils datasets methods base > > other attached packages: > [1] BSgenome.Dmelanogaster.UCSC.dm3_1.3.19 RnaSeqTutorial_0.0.11 easyRNASeq_1.4.2 > [4] ShortRead_1.16.4 latticeExtra_0.6-24 RColorBrewer_1.0-5 > [7] Rsamtools_1.10.2 DESeq_1.10.1 lattice_0.20-14 > [10] locfit_1.5-8 BSgenome_1.26.1 GenomicRanges_1.10.7 > [13] Biostrings_2.26.3 IRanges_1.16.6 edgeR_3.0.8 > [16] limma_3.14.4 biomaRt_2.14.0 Biobase_2.18.0 > [19] genomeIntervals_1.14.0 BiocGenerics_0.4.0 intervals_0.14.0 > > loaded via a namespace (and not attached): > [1] annotate_1.36.0 AnnotationDbi_1.20.7 bitops_1.0-5 DBI_0.2-5 genefilter_1.40.0 geneplotter_1.36.0 grid_2.15.1 > [8] hwriter_1.3 RCurl_1.95-4.1 RSQLite_0.11.2 splines_2.15.1 stats4_2.15.1 survival_2.37-4 tools_2.15.1 > [15] XML_3.95-0.2 xtable_1.7-1 zlibbioc_1.4.0 > > -- > Sent via the guest posting facility at bioconductor.org.