Hi Yona, Yes, the GTF file is most likely the problem due to it lacking certain attributes that Cuffdiff requires to perform these calculations. You will also want to double check that the reference genome and GTF file (where you source it next) are an exact match - both the genome build and the identifier format. If either are not a match, you will not get the expected or full results that Cuffdiff can produce. This wiki has some help; http://wiki.galaxyproject.org/Support#Interpreting_scientific_results See "Tools on the Main server: Example ? RNA-seq analysis tools." The links to the Cufflinks web site explains the attributes that Cuffdiff is looking for, links to the iGenomes datasets available (best to use if your genome is represented), and a pointer to the tool's user group. Two iGenomes GTF files are also already available in Galaxy (hg19, mm9) in "Shared Data -> Data Libraries -> iGenomes". The link to our tutorial and FAQ has help about how the GTF files are used along with troubleshooting advice. Best, Jen Galaxy team On 4/3/13 8:28 AM, Yona Kim wrote:

Dear galaxy users

Hello. I have a quick question about Cuffdiff analysis. I have obtained two SRA files and converted them to fastq files which were uploaded to Galaxy via FTP server. My analysis was followed by Fastq groomer, Tophat, Cufflinks, Cuffcompare, and eventually Cuffdiff. (Gene annotation was also downloaded from UCSC table browser in GTF format) I've downloaded gene differential expression testing, one of the output files of Cuffdiff, and viewed it in excel sheet. However, I have only zeros recorded for value_1, value_2, log2, test_stat and only ones recorded for p_value and q_value.

Is it likely that I might have obtained wrong gene annotation file and caused this problem?

Thank you

Yona Kim Department of Genetics Rutgers University - New Brunswick Campus

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