Hello Davide, The fact that you are not getting any error points to some problem with the input. Perhaps you are sending just sequence data in BAM format to Cufflinks, without any alignment performed first? Some sort of error would be expected for most other cases, but this is not the Galaxy server our team hosts, it is difficult to state exactly what the issue may be, just offer suggestions. Tophat will require fastq files as input, unless this alternate Galaxy site has a modified wrapper. Then the alignments generated by Tophat (or another alignment tool, sometimes Bowtie is used) in BAM format are the input to Cuffinks (along with other optional data). If your data are aligned BAM, and you continue to have problems with this alternate Galaxy site, it would be best to contact the group that runs it - the information is on their home page (middle panel) when you follow the url. You could also decide to use the public Galaxy instance run by our core project team at http://usegalaxy.org, if we have the tool set you wish to use. A generalized tutorial for RNA-seq analysis is available here: http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise And some troubleshooting help here: http://wiki.galaxyproject.org/Support#Tools_on_the_Main_server The tool author's original documentation would be good to review as well: http://tophat.cbcb.umd.edu http://cufflinks.cbcb.umd.edu Best, Jen Galaxy team On 12/13/12 11:47 AM, Davide Degli Esposti wrote:

Hello,

I am new in using Galaxy and I am working on .bam files generated by our sequencing platform, using the LifeScope software associated to ABI 5500 sequencer. I uploaded my files on a galaxy browser ( http://galaxy.raetschlab.org) and I tried to run cufflink assemble and quantify reads expression levels for each file. However, when I run cufflinks (using default parameters) the output is an empty file. What is going wrong? Should I use special parameters? Are the .bam files generated by LifeScope suitable for cufflink analysis or should I transform the xsq ABI output in a fastq and then apply TopHat?

I thank you very much for your help

Davide

--- Davide Degli Esposti, PhD Epigenetic (EGE) Group International Agency for Research on Cancer Tel. +33 4 72738036 Fax. +33 4 72738322 150, cours Albert Thomas 69372 Lyon Cedex 08 France

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