Hello Jianguang,

When in the analysis process to start using the reference GTF file can depend on whether or not you intend to do any discovery along with differential expression testing. At the TopHat and Cufflinks steps, using reference GTF file can influence how datasets will map and assemble. In general, if your intention is to do discovery (e.g. work with novel isoforms in your data, but not in the reference), then do not add in the reference GTF until the CuffMerge step (to produce the input annotation GTF file for Cuffdiff). But if you want to guide the analysis toward known isoforms, then use the reference GTF.


This is the process our RNA-seq example protocol follows:
http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise

For reference, there are other variations of this on the Cufflinks web site, some that never lead to Cuffdiff, but still may be useful to review. Please see the Cufflinks paper (linked from right side bar as "Protocol" for many more options/discussion.
http://cufflinks.cbcb.umd.edu/tutorial.html
--> Common uses of the Cufflinks package

The end decision will be up to you, and a few runs with different options may be a useful way to make the final call, but hopefully this provides some resources to help you understand the option,

Jen
Galaxy team

On 8/23/12 8:03 AM, Du, Jianguang wrote:
Dear All,

I am analysing RNA-seq datasets for differential splicing events between
cell types. These are mouse cells. Jen suggested me to use the iGenomes
version of reference GTF to take full advantage of the options in
CuffDiff. My question is: should I use this iGenome version reference
GTF when I run Tophat?

Thanks.

Jianguang



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