Question: Cuffdiff Error on Galaxy??
gravatar for kaybhandal
2.7 years ago by
kaybhandal0 wrote:

Hi, I am trying to run cuffdiff on Galaxy and I keep on running into the same error:

Fatal error: Exit code 1 () [11:08:04] Loading reference annotation. Error reference gene annotation differs between samples! /galaxy-repl/main/files/014/574/dataset_14574561.dat /galaxy-repl/main/files/014/535/dataset_14535943.dat: 702213666! /galaxy-

I have run this a few times and this is all I am seeing. The thing is, aside from the transcripts being GTF files and the conditions being cuffquant (cxb) files, it should have worked. My TA has given me the following instructions on how to use cuffdiff:

Run [NGS: RNA Analysis >] Select Cuffdiff. Upload the GENCODE GTF.

SQlite’ select ‘YES’. Under ‘input data type’ select ‘Cuffquant CXB’ format. For condition 1 include

the data assigned from RNA-seq assignment 2. For condition 2 include the K562 dataset.

And in the end, we are supposed to look at the ‘Gene differential FPKM’ file to identify which genes are differentially expressed between the two conditions.

I am doing everything that is listed, but it keeps on coming in error. I am not familiar with the program, so providing steps as to how I can do it would help me a lot, but any help is greatly appreciated!

Thank you!

ADD COMMENTlink modified 2.7 years ago by Jennifer Hillman Jackson25k • written 2.7 years ago by kaybhandal0
gravatar for Jennifer Hillman Jackson
2.7 years ago by
United States
Jennifer Hillman Jackson25k wrote:


Make sure that the same reference annotation was used for all steps - if used at all with these other tools (Tophat, Cufflinks, Cuffmerge - really any of tool that touched the data since it was in the original fastq format). The reference genome must also be the same one as the reference annotation was based on. In short, all must be an exact match or various problems can occur.

Mix-ups between genome data resources can be investigated. This wiki explains how to double check your data:

The tool suite manual can help with protocol:

Thanks, Jen, Galaxy team

ADD COMMENTlink modified 2.7 years ago • written 2.7 years ago by Jennifer Hillman Jackson25k
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