Hi Giuseppe, Your workflow sounds correct. Cuffmerge or Cuffcompare is needed and no, you do not want to use Cufflinks as sample input - these should be mapped reads as you state that you have used them so far. Are you using the public Main Galaxy server at http://main.g2.bx.psu.edu (usegalaxy.org)? If so and you would like to share a history, I can take a quick look to see if any usage problems are present. For the overall results - there are many factors that can contribute to different results vs what is published. It may be that parameters for some of these steps need to be tuned, or that obtaining the same results without a reference annotation to guide assembly is not possible. We can offer help torule out anything technically wrong with the workflow, so you can concentrate on these scientific questions (test/try alternate parameter settings and such). The paper from the tool authors (in the link below) would be a good place to start once you get to this point, if you are not already using it. To share, use the gear icon at the top of the history panel (upper right corner), select "Share or Publish", click on the first button to generate the share link, copy and paste that into a reply email to just me (not the entire list, to keep your data private). If instead you want to just explore this more on your own, links to help for RNA-seq tools are in this wiki: http://wiki.galaxyproject.org/Support#Interpreting_scientific_results See Tools on the Main server: Example ? RNA-seq analysis tools. Best, Jen Galaxy team On 4/16/13 7:38 AM, Ianiri, Giuseppe wrote:

Hi, I want to compare gene deferentially expressed in two conditions (1 and 2). I don't have a GTF reference annotation. In my first attempt, I mapped my Illumina reads with TopHat. Then I run Cufflink, and Cuffmerge on my Cufflink outputs for the conditions 1 and 2. At the end, I run Cuffdiff using the Cuffmerge output (1 + 2) and the TopHat output for the two samples of interest. I got genes and transcripts differentially expressed, but I was expecting a quite different output (based on the literature). Do you guys think I did anything wrong? Also, I am not sure the question makes sense, but is the Cuffmerge step essential for the comparison of my two samples? Or could I use my Cufflink outputs for samples 1 and 2 for running Cuffdiff?

Regards,

Giuseppe Ianiri Email: ianirig@umkc.edu

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