Jeremy, 

Thank you for the suggestions.  Ultimately, I would like to compare 
gene(isoform) expression between two groups of 10 animals with one lane per 
animal.  I am using the public server to practice with some small data sets 
right now, but will be getting the real data very soon and plan on using an 
Amazon Cloud account to actually do the analysis.  I can see now that this 
approach is going to be met with some difficulty with the current state of the 
data volume restrictions and limited functionality of Galaxy for 
Cuffcompare/diff.  Can you comment any further on the timeline of the 
availability of the full functionality of these programs?  You seemed to 
suggest they will be available on the public server before they are available 
on the Cloud?  

Also, for the time being, would you mind clarifying for me what you mean by 
repeatedly merging Cufflinks outputs?  I imagine using Tophat to map the reads 
and find splice junctions and  assembling transcripts using Cufflinks for each 
of the 20 animals.  Are you talking about running the Cufflinks GTF output 
through Cuffcompare, which allows two GTF files in Galaxy, and merging that 
output(the union file) with the third Cufflinks file and so on for all ten 
animals?  Then do the same thing for the other group of ten animals, and then 
comparing the two for a rough idea of the differences?  I guess I'm wondering 
how far I will be able to get with the analysis as things stand on the Cloud or 
the public server....  I also need to come up with a strategy to work around 
the 1000Gb space limit, as with 20 samples of 25 million reads and repeatedly 
generating files I think it will get used up quickly....

 As far as changing the bowtie options through Tophat, I was just going to play 
around with the bowtie mapping settings to get an idea of which strategy is 
optimal and use those settings for Tophat, but this is probably unnecessary in 
the grand scheme of the analysis.  

I really appreciate your help - Thanks,
David



-----Original Message-----
From: Jeremy Goecks on behalf of Jeremy Goecks
Sent: Tue 1/18/2011 9:29 PM
To: Martin, David A.
Cc: eaf...@emory.edu; galaxy-u...@bx.psu.edu
Subject: Re: [galaxy-user] Galaxy for gene expression comparison
 

> I am comparing RNA expression in two groups of rats, a drug treated group 
> against a control group. There are 10 biological replicates in each group.  I 
> am unsure of how to flow this analysis through Galaxy using Tophat followed 
> by Cufflinks/compare/diff. Should the files for each group be merged at any 
> point?  I would think they should be kept separate in order to properly 
> account for the spread across animals.  I am just a little unsure of how to 
> group the files on galaxy, and where to differentiate biological and 
> technical replicates.
> 
Hi David,

Yes, you're right -- merging along the way will prevent you from quantifying 
within-group variation; consequently, quantifying across-group variation will 
be very challenging as well. Here's the right thing to do:

(1) map each replicate using Tophat and assemble transcripts using Cufflinks;
(2) for all Cufflinks' outputs (assembled transcripts), build a set of 
comprehensive transcripts using Cuffcompare;
(3) for Cuffdiff, group the replicates from each group and let Cuffdiff 
determine and quantitate within group and across group variation.

However, Galaxy's tools currently don't support replicates, so you can't yet 
perform this analysis. We're working to enhance them, however, and we should 
have this functionality available on our main server in the next couple weeks. 
Enis can comment about when this functionality will be available on the cloud.

(To be clear, you can perform step 1 using Galaxy now. You can also perform 
step 2, but you'll have to do so by repeatedly merging Cufflinks' outputs. You 
cannot perform step 3 right now with Galaxy.)
> On a different note, is there a way to control the bowtie mapping parameters 
> more closely when using tophat?
> 
There's limited control that you can exert over the bowtie commands within 
Tophat. Looking at the Tophat manual:

http://tophat.cbcb.umd.edu/manual.html

it looks like max-multihits (Maximum number of alignments) is the only Bowtie 
parameter you can directly control. There are, however, many Tophat parameters 
that enable you to control splice junction mapping directly; set Tophat's 
settings to 'Full parameter list' to see all the parameters you can control. 
What exactly are you looking to do?

Thanks,
J.

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