Hello Wei, The results do sound strange. The best advice to start with is to make sure that you are up-to-date with both Galaxy and the RNA-seq tools and using the best possible inputs. 1 . Make sure that you are running the latest distribution http://wiki.galaxyproject.org/DevNewsBriefs 2. Update to use the current version of CuffDiff http://wiki.galaxyproject.org/Admin/Tools/Tool%20Dependencies 3. Use the iGenomes GTF annotation to make full use of the functionality in Cuffdiff http://cufflinks.cbcb.umd.edu/igenomes.html A good test to see if your set-up is correct would be to run the RNA-seq tutorial locally as a test case. http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise The reverse can also be done, if you have a problem locally, try running it as a small test (that still demonstrates the issue) on the Public Main server and see if the results can be duplicated. This can help determine if problem is with data inputs/settings or a problem with tool set-up/installation. It can also be a way to share your data with us if you need feedback. But, hopefully after updating the issue clears up! Jen Galaxy team On 11/29/12 11:51 AM, Wei Liao wrote:

Hi, Galaxy user. I ran into a problem when using Cuffdiff 1.2.1 in Galaxy local instance to check differential expressed genes in my samples. I have 3 normals and 6 cancers samples, I did the following: - After tophat for each samples, run cufflink with refseq annotation which has 25266 genes and 43091 transcripts - cuffmerge all cufflink outputs contains 58112 lines - run cuffdiff with 3 normals as triplicate and compare to each cancer sample. Suprisingly, I fould out that the tanscripts tracking file, gene tracking, CDS tracking only has 2000 genes and 4000 transcripts. So the cufflink only compare 2000 genes and 4000 transcripts between samples. The question I want to ask here is that *why are the rest of the genes and transcripts not being tested and included in the tracking files?* Do you know what cause this kind of problem? Thanks, Wei

-- Wei Liao Research Scientist, Brentwood Biomedical Research Institute 16111 Plummer St. Bldg 7, Rm D-122 North Hills, CA 91343 818-891-7711 ext 7645

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