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.