Dear Bioconductor Team, after I running TopHat - Cufflinks - Cuffmerge - Cuffdiff and try to create plots with cummeRbund I have trouble with importing my data. I follow protocol from Nature (Differential gene transcripts expression analysis of RNA-seq experiment with TopHat and Cufflinks). I use gtf file downloaded from iGenomes. When I wants plot some basic graphs it is work - csDensity,csBoxplot..But some doesnt work - expressionBarplot,fpkmSCVPlot... I am paste down my codes. I found on internet the same issue but without any good solutions. Thank you so much, Petr. -- output of sessionInfo(): > cuff <- readCufflinks() Creating database /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_sam ples_diff/cuffData.db Reading Run Info File /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_diff/run.info Writing runInfo Table Reading Read Group Info /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/ all_samples_diff/read_groups.info Writing replicates Table Reading /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_diff/ genes.fpkm_tracking Checking samples table... Populating samples table... Writing genes table Reshaping geneData table Recasting Writing geneData table Reading /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_diff/ gene_exp.diff Error in (function (classes, fdef, mtable) : unable to find an inherited method for function ???make.db.names??? for signature ???"SQLiteConnection", "integer"??? > cuff Error: object 'cuff' not found > cuff <- readCufflinks() dir= gtfFile= repTableFile= geneDiff= geneRep= isoformDiff= isoformRep= TSSDiff= TSSRep= CDSExpDiff= CDSRep= promoterFile= driver= rebuild= ...= dbFile= runInfoFile= geneFPKM= geneCount= isoformFPKM= isoformCount= TSSFPKM= TSSCount= CDSFPKM= CDSCount= CDSDiff= splicingFile= genome= verbose= > cuff <- readCufflinks() > cuff CuffSet instance with: 4 samples 23235 genes 0 isoforms 0 TSS 0 CDS 0 promoters 0 splicing 0 relCDS -- Sent via the guest posting facility at bioconductor.org.

Hi Petr, Please try keep threads on the bioconductor mailing list so that others may benefit from the answers provided. The issue here is that your sample names are integers and R will not allow integers to be used as column names in a data.frame. The easiest solution is to re-run your cuffdiff analysis using labels for your samples that do not start with a numeric value (0-9). Best of luck! -Loyal On May 7, 2014, at 10:31 AM, Petr Broz <bbrozpetr@gmail.com> wrote: > The naming was just numbers - 7510,7511,7512,7877. > > Petr. > > > On Wed, May 7, 2014 at 4:21 PM, Petr Broz <bbrozpetr@gmail.com> wrote: > Dear Loyal, > > thank you for very fast response. I used newest Tophat - 2.0.11 and for cufflinks and cuffmerge 2.2.0 and for cuffdiff 2.2.1 (I found newest version just today of cufflinks). But I had the absolutely same problem when I used the version cufflinks 2.2.0 for all analysis one week ago. > > Here is session info: > > > sessionInfo() > R version 3.1.0 (2014-04-10) > Platform: x86_64-pc-linux-gnu (64-bit) > > locale: > [1] LC_CTYPE=en_GB.UTF-8 LC_NUMERIC=C > [3] LC_TIME=en_GB.UTF-8 LC_COLLATE=en_GB.UTF-8 > [5] LC_MONETARY=en_GB.UTF-8 LC_MESSAGES=en_GB.UTF-8 > [7] LC_PAPER=en_GB.UTF-8 LC_NAME=C > [9] LC_ADDRESS=C LC_TELEPHONE=C > [11] LC_MEASUREMENT=en_GB.UTF-8 LC_IDENTIFICATION=C > > attached base packages: > [1] grid parallel stats graphics grDevices utils datasets > [8] methods base > > other attached packages: > [1] cummeRbund_2.5.3 Gviz_1.7.10 rtracklayer_1.23.22 > [4] GenomicRanges_1.15.44 GenomeInfoDb_0.99.30 IRanges_1.21.44 > [7] fastcluster_1.1.13 reshape2_1.2.2 ggplot2_0.9.3.1 > [10] RSQLite_0.11.4 DBI_0.2-7 BiocGenerics_0.9.3 > > loaded via a namespace (and not attached): > [1] AnnotationDbi_1.25.17 BatchJobs_1.2 > [3] BBmisc_1.5 Biobase_2.23.6 > [5] BiocParallel_0.5.20 biomaRt_2.19.3 > [7] Biostrings_2.31.22 biovizBase_1.11.15 > [9] bitops_1.0-6 brew_1.0-6 > [11] BSgenome_1.31.13 cluster_1.15.2 > [13] codetools_0.2-8 colorspace_1.2-4 > [15] dichromat_2.0-0 digest_0.6.4 > [17] fail_1.2 foreach_1.4.1 > [19] Formula_1.1-1 GenomicAlignments_0.99.38 > [21] GenomicFeatures_1.15.15 gtable_0.1.2 > [23] Hmisc_3.14-3 iterators_1.0.6 > [25] labeling_0.2 lattice_0.20-29 > [27] latticeExtra_0.6-26 MASS_7.3-31 > [29] matrixStats_0.8.14 munsell_0.4.2 > [31] plyr_1.8.1 proto_0.3-10 > [33] RColorBrewer_1.0-5 Rcpp_0.11.1 > [35] RCurl_1.95-4.1 R.methodsS3_1.6.1 > [37] Rsamtools_1.15.41 scales_0.2.3 > [39] sendmailR_1.1-2 splines_3.1.0 > [41] stats4_3.1.0 stringr_0.6.2 > [43] survival_2.37-7 tools_3.1.0 > [45] VariantAnnotation_1.9.49 XML_3.98-1.1 > [47] XVector_0.3.7 zlibbioc_1.9.0 > > Thank you for help, > > Petr. > > > On Wed, May 7, 2014 at 4:07 PM, Loyal A. Goff <lgoff@csail.mit.edu> wrote: > Also, > Can you tell me what the sample names were in your experiment? It may be relevant to why the DB build failed. > > Loyal > > Sent from my iPad > > > On May 7, 2014, at 8:44 AM, Petr [guest] <guest@bioconductor.org> wrote: > > > > > > Dear Bioconductor Team, > > > > after I running TopHat - Cufflinks - Cuffmerge - Cuffdiff and try to create plots with cummeRbund I have trouble with importing my data. I follow protocol from Nature (Differential gene transcripts expression analysis of RNA-seq experiment with TopHat and Cufflinks). I use gtf file downloaded from iGenomes. When I wants plot some basic graphs it is work - csDensity,csBoxplot..But some doesnt work - expressionBarplot,fpkmSCVPlot... I am paste down my codes. > > > > I found on internet the same issue but without any good solutions. > > > > Thank you so much, > > > > Petr. > > > > -- output of sessionInfo(): > > > >> cuff <- readCufflinks() > > Creating database /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all _samples_diff/cuffData.db > > Reading Run Info File /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_diff/run.info > > Writing runInfo Table > > Reading Read Group Info /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_ rna/all_samples_diff/read_groups.info > > Writing replicates Table > > Reading /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_d iff/genes.fpkm_tracking > > Checking samples table... > > Populating samples table... > > Writing genes table > > Reshaping geneData table > > Recasting > > Writing geneData table > > Reading /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_d iff/gene_exp.diff > > Error in (function (classes, fdef, mtable) : > > unable to find an inherited method for function ‘make.db.names’ for signature ‘"SQLiteConnection", "integer"’ > >> cuff > > Error: object 'cuff' not found > >> cuff <- readCufflinks() > > dir= gtfFile= repTableFile= geneDiff= geneRep= isoformDiff= isoformRep= TSSDiff= TSSRep= CDSExpDiff= CDSRep= promoterFile= driver= rebuild= ...= > > dbFile= runInfoFile= geneFPKM= geneCount= isoformFPKM= isoformCount= TSSFPKM= TSSCount= CDSFPKM= CDSCount= CDSDiff= splicingFile= genome= verbose= > >> cuff <- readCufflinks() > >> cuff > > CuffSet instance with: > > 4 samples > > 23235 genes > > 0 isoforms > > 0 TSS > > 0 CDS > > 0 promoters > > 0 splicing > > 0 relCDS > > > > > > -- > > Sent via the guest posting facility at bioconductor.org. > > [[alternative HTML version deleted]]

Hi petr, Your cummerbund database is not fully built ( as indicated by the first 'error' in your pasted code), but the database was initialized and at least one table populated. Subsequent calls to readCufflinks are establishing connections to the malformed database, but the data required to generated the plots have not been incorporated. First things first, we need to figure out why your database did not build correctly. Can you please paste the results of a call to sessionInfo(), and elaborate on what version of cufflinks/cuff diff was used to process your reads? Loyal Sent from my iPad > On May 7, 2014, at 8:44 AM, Petr [guest] <guest at="" bioconductor.org=""> wrote: > > > Dear Bioconductor Team, > > after I running TopHat - Cufflinks - Cuffmerge - Cuffdiff and try to create plots with cummeRbund I have trouble with importing my data. I follow protocol from Nature (Differential gene transcripts expression analysis of RNA-seq experiment with TopHat and Cufflinks). I use gtf file downloaded from iGenomes. When I wants plot some basic graphs it is work - csDensity,csBoxplot..But some doesnt work - expressionBarplot,fpkmSCVPlot... I am paste down my codes. > > I found on internet the same issue but without any good solutions. > > Thank you so much, > > Petr. > > -- output of sessionInfo(): > >> cuff <- readCufflinks() > Creating database /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_s amples_diff/cuffData.db > Reading Run Info File /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_diff/run.info > Writing runInfo Table > Reading Read Group Info /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rn a/all_samples_diff/read_groups.info > Writing replicates Table > Reading /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_dif f/genes.fpkm_tracking > Checking samples table... > Populating samples table... > Writing genes table > Reshaping geneData table > Recasting > Writing geneData table > Reading /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_dif f/gene_exp.diff > Error in (function (classes, fdef, mtable) : > unable to find an inherited method for function ?make.db.names? for signature ?"SQLiteConnection", "integer"? >> cuff > Error: object 'cuff' not found >> cuff <- readCufflinks() > dir= gtfFile= repTableFile= geneDiff= geneRep= isoformDiff= isoformRep= TSSDiff= TSSRep= CDSExpDiff= CDSRep= promoterFile= driver= rebuild= ...= > dbFile= runInfoFile= geneFPKM= geneCount= isoformFPKM= isoformCount= TSSFPKM= TSSCount= CDSFPKM= CDSCount= CDSDiff= splicingFile= genome= verbose= >> cuff <- readCufflinks() >> cuff > CuffSet instance with: > 4 samples > 23235 genes > 0 isoforms > 0 TSS > 0 CDS > 0 promoters > 0 splicing > 0 relCDS > > > -- > Sent via the guest posting facility at bioconductor.org.

Also, Can you tell me what the sample names were in your experiment? It may be relevant to why the DB build failed. Loyal Sent from my iPad > On May 7, 2014, at 8:44 AM, Petr [guest] <guest at="" bioconductor.org=""> wrote: > > > Dear Bioconductor Team, > > after I running TopHat - Cufflinks - Cuffmerge - Cuffdiff and try to create plots with cummeRbund I have trouble with importing my data. I follow protocol from Nature (Differential gene transcripts expression analysis of RNA-seq experiment with TopHat and Cufflinks). I use gtf file downloaded from iGenomes. When I wants plot some basic graphs it is work - csDensity,csBoxplot..But some doesnt work - expressionBarplot,fpkmSCVPlot... I am paste down my codes. > > I found on internet the same issue but without any good solutions. > > Thank you so much, > > Petr. > > -- output of sessionInfo(): > >> cuff <- readCufflinks() > Creating database /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_s amples_diff/cuffData.db > Reading Run Info File /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_diff/run.info > Writing runInfo Table > Reading Read Group Info /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rn a/all_samples_diff/read_groups.info > Writing replicates Table > Reading /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_dif f/genes.fpkm_tracking > Checking samples table... > Populating samples table... > Writing genes table > Reshaping geneData table > Recasting > Writing geneData table > Reading /home/iab/CUSTOMERS/HiSeq/Dr.Susor/mouse_rna/all_samples_dif f/gene_exp.diff > Error in (function (classes, fdef, mtable) : > unable to find an inherited method for function ?make.db.names? for signature ?"SQLiteConnection", "integer"? >> cuff > Error: object 'cuff' not found >> cuff <- readCufflinks() > dir= gtfFile= repTableFile= geneDiff= geneRep= isoformDiff= isoformRep= TSSDiff= TSSRep= CDSExpDiff= CDSRep= promoterFile= driver= rebuild= ...= > dbFile= runInfoFile= geneFPKM= geneCount= isoformFPKM= isoformCount= TSSFPKM= TSSCount= CDSFPKM= CDSCount= CDSDiff= splicingFile= genome= verbose= >> cuff <- readCufflinks() >> cuff > CuffSet instance with: > 4 samples > 23235 genes > 0 isoforms > 0 TSS > 0 CDS > 0 promoters > 0 splicing > 0 relCDS > > > -- > Sent via the guest posting facility at bioconductor.org.