> 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:


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?

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