Thanks for your help.
Do you mean that if I want to find novel isoform/splicing, I need to select
"No" under "Use Reference Annotation" when I run Cufflink, and then use iGenome
version of reference GTF when I run Cuffmerge?
Based on your information and some protocols found online, my understanding is
1) if use iGenome version of reference GTF, I only need to run Cuffmerge with
the Cufflink ouputs, because iGenome version reference GTF already contains
attributes such as p_id and tss_id. Then the Cuffmerge output can be used for
2) however, if I use the reference GTF from Ensembl/UCSC (rather than from
iGenome), I need to run Cuffcompare to create p_id and tss_id, which is
required for Cuffdiff.
Am I right?
Another question is: should I use iGenome version of reference GTF when I run
Tophat if I want to see novel isoforms/splicing?
From: Jennifer Jackson [j...@bx.psu.edu]
Sent: Thursday, August 23, 2012 11:46 AM
To: Du, Jianguang
Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for
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:
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.
--> 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,
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?
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The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org. Please keep all replies on the list by
using "reply all" in your mail client. For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:
To manage your subscriptions to this and other Galaxy lists,
please use the interface at: