Hi Jen,
I had a problem when I tried to run Tophat with the iGenome reference GTF.
What I did is:
1) uploaded iGenome version of mm9 genes.gtf by: Shared Data -> Data Libraries -> iGenomes -> click "genes.gtf"
under "mm9" -> click "Go" for "Import to current history". The genes.gtf appeared in history and
turned green.
2) click "Tophat for Illumina Find splice junctions using RNA-seq data" to open the
window of "Tophat for Illumina (version 1.5.0)"
3) selected the dataset to be analysed under "RNA-Seq FASTQ file:".
4) chose "Use one from the history" under "Will you select a reference genome from
your history or use a built-in index?:"
Then the screen refreshed and the box (pulldown menu) under "Select the reference
genome:" became smaller. Nothing showed up in the pulldown menu (actually the menu can not be
pulled down). So that I could not input iGenome reference GTF. Looks like the Tophat can only
"Use a built-in index".
How can I solve this problem?
Thanks in advance.
Jianguang
________________________________________
From: galaxy-user-boun...@lists.bx.psu.edu
[galaxy-user-boun...@lists.bx.psu.edu] on behalf of Du, Jianguang
[jia...@iupui.edu]
Sent: Thursday, August 23, 2012 4:01 PM
To: Jennifer Jackson
Cc: galaxy-user@lists.bx.psu.edu
Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for
Tophat?
Hi Jen,
Thank you very much for your help.
Jianguang
________________________________________
From: Jennifer Jackson [j...@bx.psu.edu]
Sent: Thursday, August 23, 2012 3:53 PM
To: Du, Jianguang
Cc: galaxy-user@lists.bx.psu.edu
Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for
Tophat?
Hello Jianguang,
On 8/23/12 11:28 AM, Du, Jianguang wrote:
Hi Jen,
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?
Yes, according to the tool documentation, this is the method.
Based on your information and some protocols found online, my understanding is
that:
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
Cuffdiff.
Yes, this is the example protocol I shared.
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.
This can be tricky, it depends on what order you run the tools with and
without the GTF annotation. The protocol in #1 is recommended.
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?
Yes, this is what I intended to answer in my original reply, I apologize
if that was not clear. The reference GTF can influence both mapping and
assembly. So, both Tophat and Cufflinks. The information on the TopHat
web site for the parameter provides more information (see link on TopHat
tool form). The tool authors can also be contacted if there are some
details that you are curious about that are not covered in the primary
documentation: tophat.cuffli...@gmail.com
Others are welcome to add to the thread with their experiences if they
have used a reference annotation GTF with Tophat (or chosen not to for a
particular reason that they would like to share),
Best,
Jen
Galaxy team
Thanks.
Jianguang
________________________________________
From: Jennifer Jackson [j...@bx.psu.edu]
Sent: Thursday, August 23, 2012 11:46 AM
To: Du, Jianguang
Cc: galaxy-user@lists.bx.psu.edu
Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for
Tophat?
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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Jennifer Jackson
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--
Jennifer Jackson
http://galaxyproject.org
___________________________________________________________
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
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use the Galaxy Development list:
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