Thank you very much for your reply. For the time being, I just wanted to be familiarized with the workflow and the open resource of galaxy main to analyze NGS. If you could advise me how I can obtain the RPKM that will be great. I have tried many ways to map but no luck so far. I think I’m at the end of my wits.

I did try tophat2 with ion torrent data and it worked fine. This solid SRA format is giving me a hard time and I can only work on webbase program. I also try tophat for solid on test server but it failed! Many thanks again

Best regards

Dao

From: Jennifer Jackson [mailto:jen@bx.psu.edu]
Sent: November 20, 2013 8:46 PM
To: Ly, Dao; galaxy-user@lists.bx.psu.edu
Subject: Re: [galaxy-user] Cufflinks returned 0 value in all RPKMs

Hello,

If the data is RNA from rat, then you will want to be using Tophat instead of Bowtie. Otherwise the data will not be mapped as spliced the results will be off in many ways (the fragments counts are a small symptom of a larger problem).

You can use 'Tophat for SOLiD' on a suitable local or cloud Galaxy instance. It is available on the Test server, but tools are not supported here (we test/break things!) and the quotas are just 10G with an account. But maybe is a place to do a small trial run before committing to a cloud server.
http://getgalaxy.org
http://usegalaxy.org/cloud
http://usegalaxy.org/toolshed

More about RNA-seq is in our wiki and public server, including link-outs and tutorials, you can get started here:
Example → RNA-seq analysis tools: http://wiki.galaxyproject.org/Support#Interpreting_scientific_results
See RNA-seq examples: http://wiki.galaxyproject.org/Learn#Other_Tutorials

Best,

Jen
Galaxy team

On 11/20/13 6:09 AM, Ly, Dao wrote:

Hi

I have been trying to analyze a rat Solid SRA but I encountered a problem: cufflinks gave me 0 RPKM in all genes. Here is my workflow

1.       Get data with EBI SRA: sent the fastaq file directly to galaxy

2.       Fastaq groomer

3.       Mapped with bowtie for Solid (paire-ended) with the built- in index rat rn5 as reference genome

4.       Sam to Bam the bowtie mapping result

5.       Cufflinks the bam file

All RPKMs of gene expression and transcript expression have a 0 value even thought the RPKM status is OK. I used default setting for all jobs. Am I missing something? Any help, suggestion will be greatly appreciated. Thank you very much

Best regards

Dao
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