I've been having trouble running my Arabidopsis thaliana NGS pipeline
in galaxy. Specifically, tophat alignment to the built-in-index worked
fine (visual assessment in IGV) but I couldn't get cufflinks/Cuffduiff
to run using the same annotation.gtf file I'd used in the alignment.
Error message = Error running cufflinks. return code = -11 cufflinks:
Cufflinks/cuffdiff would run without the annotation file and give a
differential expression output but without the annotation file the
gene_id was meaningless (XLOC_000001 etc) and all I had was a locus
for a region with differential expression.
After a lot of frustration I re ran the alignment using a custom built
genome and annotation file from Illumina:
http://cufflinks.cbcb.umd.edu/igenomes.html and hey presto,
cufflinks/cuffdiff work fine. I did try altering using both chr1 and 1
for chromosome name as a discord between the chromosome naming seemed
to frequently be a problem. Apart from this, what other reasons could
there be for the built-in-index not working with annotation.gtf files?
Is this an issue with the built-in-index (Mouse-ear Cress (Arabidopsis
Tom SmithUniversity of York, UK
A mismatch between chromosome identifiers can definitely be
These have to be exact.
The TAIR 10 genome in Galaxy has these identifiers:
Comparing these to your GTF file would be the way to see if any
It is curious GTF file worked with Tophat, but then not with
Cufflinks/Cuffdiff. Did you really use the Junction/Gene Annotation
model option, and the GTF file worked OK with TopHat, then failed
with Cufflinks/diff? This may mean that the problem is not with the
file at all, but rather with something else in the method/settings.
it work with Cuffcompare or Cuffmerge?
There are no known issues with the index and it is the same index
used for all tools (Bowtie/Tophat/Cufflinks-merge-compare-diff), but
you continue to have problems after double checking the IDs and other
methods (steps between Tophat and Cufflinks, if any, then steps
Cufflinks and Cuffdiff), please send in a bug report and we can try to
Just for reference, this is our tutorial for RNA-seq analysis:
Hopefully this helps, but if this does not fully resolve the issue, we
can work together through the bug report,