Dear Sir/Madam, I am a postdoctoral researcher at Chris Mason's lab at the ICB Cornell Medical College in NYC. I would be very interested in using your R package for RNA-seq analysis of some raw data we have. We went through the manual quickly, and it is not clear for me how to start the analysis, i.e., input file. To be more specific: given the fastq file (or binary fastq.tbz), could we use it as input for Voom, and then use the result for Limma? Or should we align first our raw fastq data and then use the sam or bam files as input for the Vomm or Limma packages? How should I proceed to start an analysis from raw fastq files? I would highly appreciate an answer at your earliest convenience. Thank you very much in advance for your attention, -- Best, Pedro Pedro Blecua, Ph.D. Postdoctoral Associate Weill Medical College of Cornell University Institute for Computational Biomedicine Department of Physiology and Biophysics 1305 York Avenue, Box 140, New York, NY 10021 Office: 212 746 4237 Fax: 212 746 8690

Hi Pedro, On 1/7/2013 3:47 PM, Pedro Blecua wrote: > Dear Sir/Madam, > > I am a postdoctoral researcher at Chris Mason's lab at the ICB Cornell > Medical College in NYC. > I would be very interested in using your R package for RNA-seq > analysis of some raw data we have. > We went through the manual quickly, and it is not clear for me how to > start the analysis, i.e., input file. > > To be more specific: given the fastq file (or binary fastq.tbz), could > we use it as input for Voom, and then > use the result for Limma? Or should we align first our raw fastq data > and then use the sam or bam files as > input for the Vomm or Limma packages? How should I proceed to start an > analysis from raw fastq files? You need to align using a gapped aligner (bowtie2, gsnap, etc), and then use the resulting bam file to get counts per transcript, which is the input to voom. Once you have the aligned data, you can use GenomicFeatures and the correct transcript.db package to get the counts using summarizeOverlaps(). Given aligned bam files, I usually do something like library(Rsamtools) library(GenomicFeatures) bflst <- BamFileList(<character vector="" of="" bam="" files,="" including="" path="" if="" not="" in="" working="" dir="">) library(Tx.Db.Hsapiens.UCSC.hg19.knownGene) ## substitute applicable species here feat <- exonsBy(Tx.Db.Hsapiens.UCSC.hg19.knownGene, by = "gene") olaps <- summarizeOverlaps(feat, bflst) then you can do counts <- assays(olaps)$counts voom(counts) Best, Jim > > I would highly appreciate an answer at your earliest convenience. > > Thank you very much in advance for your attention, > > -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099

Dear Pedro, You'll need to align your fastq data, summarize read counts to genes and then run limma voom or edgeR for expression analysis. We have developed a pipeline for doing this which allows you to complete the entire analysis in R. This pipeline starts with using Rsubread package for alignment (see Rsubread vignette for more details, but briefly the functions 'buildindex' and 'align' are used for index building and read mapping, respectively), and then uses featureCounts function in the same package to do the read summarization. The summarized read counts can then be fed into limma or edgeR for expression analysis. Cheers, Wei On Jan 8, 2013, at 7:47 AM, Pedro Blecua wrote: > Dear Sir/Madam, > > I am a postdoctoral researcher at Chris Mason's lab at the ICB Cornell > Medical College in NYC. > I would be very interested in using your R package for RNA-seq > analysis of some raw data we have. > We went through the manual quickly, and it is not clear for me how to > start the analysis, i.e., input file. > > To be more specific: given the fastq file (or binary fastq.tbz), could > we use it as input for Voom, and then > use the result for Limma? Or should we align first our raw fastq data > and then use the sam or bam files as > input for the Vomm or Limma packages? How should I proceed to start an > analysis from raw fastq files? > > I would highly appreciate an answer at your earliest convenience. > > Thank you very much in advance for your attention, > > > -- > Best, > > Pedro > > Pedro Blecua, Ph.D. > Postdoctoral Associate > Weill Medical College of Cornell University > Institute for Computational Biomedicine > Department of Physiology and Biophysics > 1305 York Avenue, Box 140, > New York, NY 10021 > > Office: 212 746 4237 > Fax: 212 746 8690 > > _______________________________________________ > 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 ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:6}}