Question About Using Replicate Samples And Refrence Ensembl

Question: Question About Using Replicate Samples And Refrence Ensembl

7.9 years ago by

vasu punj • 360

vasu punj • 360 wrote:

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 donât see any option in Galaxy? Any suggestion please. Â

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modified 7.9 years ago by Jeremy Goecks • 2.2k • written 7.9 years ago by vasu punj • 360

7.9 years ago by

Jeremy Goecks • 2.2k

Jeremy Goecks • 2.2k wrote:

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.

ADD COMMENT • link written 7.9 years ago by Jeremy Goecks • 2.2k

7.9 years ago by

Jeremy Goecks • 2.2k

Jeremy Goecks • 2.2k wrote:

Hi Vasu, Please cc the galaxy-user email list so that everyone can benefit from this discussion and so that it is archived. On to your questions: The answer depends on what you're looking for. There are two options: (1) If you're looking for differences between the two samples--e.g. the samples came from two different tissues or from two different time periods in the same tissue--you should run each sample through Tophat & Cufflinks and then run Cuffcompare and Cuffdiff on the GTF files generated by Cufflinks. (2) If you're looking for differences within the two samples--e.g. the samples are two lanes of sequencing data from the same biological replicate--then you should combine all your reads before running Tophat-->Cufflinks-->Cuffcompare. The Ensemble gene annotation GTF should be used as the "reference" for Cuffcompare; optionally, you can also use the GTF as a reference for Cufflinks as well. Best, J.

ADD COMMENT • link written 7.9 years ago by Jeremy Goecks • 2.2k

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