Hi Meike,

Sorry for the delay. The Cuffdiff manual (http://cufflinks.cbcb.umd.edu) has help for interpreting "NO TEST/LOW DATA" results, and for adjusting the "-c option" - these are most often related to low coverage and/or fragmented transcripts.

I am wondering, did you run Cufflinks to assemble the data first? This wiki section has help for recommended protocols (and includes links back to the tool's site above):
https://wiki.galaxyproject.org/Support#Interpreting_scientific_results
See -> Tools on the Main server: RNA-seq

As explained, it could be that there is a mismatch between the reference annotation file and your mapped data. The gtf file contains chromosome identifiers using Ensembl's nomenclature, while the built-in reference genome used for mapping "rn5" is sourced from UCSC and uses their nomenclature. These are formatted differently. An exact match is required between identifiers or the annotation will be effectively be ignored. Often adding a "chr" to the start of the Ensembl chromosome name will resolve the match, but not always. The source for "rn5" was here: http://hgdownload.cse.ucsc.edu/goldenPath/rn5/bigZips/

iGenomes is the preferred reference annotation source, due to the inclusion of all the attributes specifically used by the Cuffdiff tool and how these are created with specific data sources in mind (adjusted for their identifier nomenclature). This build is not yet available, but rn4 is: http://cufflinks.cbcb.umd.edu/igenomes.html

Checking protocol and that the identifiers are a match are the first steps. Examine parameter tuning after.

I didn't find this data in any of the histories you already shared, but the above help will resolve/explain most issues or results from this pipeline.

Best,

Jen
Galaxy team

On 4/8/14 5:46 AM, Meike.Latz@mdc-berlin.de wrote:

Dear all,

I have Illumina RNAseq data and want to look for differences in gene expression between male and female rats and transgenic vs. wildtype; for each condition I have triplicates. I mapped with TopHat for Illumina, using the reference genome rn5 and default settings. I did Cuffdiff afterwards and used the GTF-file Rattus norvegicus.Rnor_5.0.72.gtf as transcript. As result I got no significant changes in expression and it always says "NO TEST" (or sometimes "LOW DATA"). I found that lowering or raising the -c option might control the cuffdiff behavior, but I do not know what it is and how to control it. Can you explain me how to do it? And would you advise me to use another transcript for Cuffdiff? And when yes, where can I get it?

Lots of questions. Thanks in advance for your answer.

Best,
Meike
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