Dear Jeremy,

Thank you for your advice!

However when I tried this out I got some more questions.

I am dealing with a bacterium which has about 4000 genes. When I tried
Cuffmerge to merge everything with reference annotation, I got a merged
file of only 50 lines. If I left out the reference annotation file,
Cuffmerge returned me a merged file of 4000 lines (which is more

However this difference didn't happen if I use Cuffcompare to merge all the
files. With or Without reference annotation, the merged file are both of
4000 lines. If I continue to Cuffdiff with this Cuffcompare file, I got
over 1000 significantly changed genes.

Could you give me some suggestion on this? Should I just trust the
Cuffcompare file?

Is it possible that there might be some problem with my reference
annotation file?

Thank you very much,


On Sun, Mar 3, 2013 at 9:59 AM, Jeremy Goecks <>wrote:

> > My question is, if I need to compare between 5 time points, should I do
> comparison pairwise?
> No, do them all at once with Cuffdiff:
> (a) set 'Perform Replicate Analysis' to 'Yes';
> (b) create 5 replicate conditions, one for each time point;
> (c) add your replicates for each time point.
> There's a Cuffdiff flag to do time series analysis, but it isn't
> implemented yet in Galaxy, so you'll get pairwise comparisons for all
> conditions. You can use the filtering tool to reduce Cuffdiff outputs to
> only the timepoint comparisons.
> > I will use cuffmerge to merge 0hour-1, 0hour-2, 0hour-3,
> 1hour-1,1hour-2.1hour-3 to generate one cuffmerge file.
> Correct.
> > Then I will run cuffdiff using the merged file, include two groups,
> group 1 is 0 hour (add 0hour 1-3 in group 1) and group 2 is 1hour (add
> 1hour1-3 in group 2).
> Use the process I described above to do all pairwise comparisons in one
> run.
> Good luck,
> J.

Qian Dong
Bauer Lab, MCBD
Simon Hall: 313-317
212 S. Hawthorne Dr.
Bloomington, IN 47405
Lab Phone:812-855-8443
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