I am running RNA seq and while I have to run Tophat how can I include replicates of samples than secondly I need to include Ensembl reference database for getting IDs in Cufflinker I don’t see any option in Galaxy? Any suggestion please.

It's not clear what you mean by "include replicates" when performing analyses via Tophat. One option is to concatenate all your reads into a single file and run Tophat on that file. Can you be more specific about your question?

You can get an Ensembl gene annotation in GTF format for cufflinks by taking the following steps in Galaxy:

(a) in Galaxy tools, go to Get Data --> UCSC Main (b) select clade/genome/assembly (c) for group, use 'Genes and Gene Prediction Tracks' (d) for track, use Ensembl (e) for table, choose the default (enGene) (f) region: genome (g) output format: GTF (h) make sure 'Send to Galaxy' is checked

Thanks, J.