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Old 11-19-2012, 08:32 PM   #21
Peppe
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Thanks jgoecks,
Yes, I am using the public Galaxy server.
As you said the job took a while to start, but after that it was quick enough. Now there is an update of the server so I have to wait. After that I will go ahead with Cufflinks,
Cuffcompare, Cuffmerge and Cuffdiff. Still not sure what it comes out and how to handle the data, but I will figure out. This is the first time that I am going through RNAseq analysis and having a forum like this is very helpful.
Thanks a lot,
Peppe
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Old 11-19-2012, 09:34 PM   #22
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Hello,
I tried to run cufflinks after TopHat mapping and as a genome reference annotation it requires a file in gtf format. I downloaded the genome annotation from the JGI website (I am working with a fungus whose genome was sequenced in 2010). The file annotation that I have is in gtf, but when I extract it it becomes gff, a format not supported by cufflinks in Galaxy. I converted my file with the workflow found at https://main.g2.bx.psu.edu/u/jeremy/...with-cufflinks. The generated genome annotation is in the gtf format (the only difference that I see is in the first column, scaffold become chrscaffold), but when I run cufflinks all the output were 0. Now I am running again cufflinks without using the annotated genome to see if there is any difference in the results. I was wondering if the conversion that I did is correct, if anyone knows if there is another way to do it, or if I should email JGI people to see if they have a GTF format for the genome annotation I need. I don't think I can go ahead with the analysis without a genome annotation in GTF.
Thanks
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Old 11-26-2012, 04:30 PM   #23
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As you guys can see in the previous messages, I am new in this field. I am using the public Galaxy server, and for the first time I run sample with TopHat-Cufflink-Cuffcompare-Cuffdiff. Can anyone tell me how to get the data generated by Cuffdiff? Is there any tool in Galaxy for visualizing them? I am interested in gene/transcript differential expression testing. Should I select only the genes with significance YES?
Thank you
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Old 11-28-2012, 03:22 PM   #24
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@Peppe

Cummerbund is the preferred option for visualizing Cuffdiff output, but it's not yet integrated into Galaxy.

However, there is a scatterplot that's handy for visualizing FPKM data for different conditions: click on the visualize icon at the bottom of a Cuffdiff expression dataset and plot column 10 (sample 1 FPKM) vs. column 14 (sample 2 FPKM). And, yes, you may want to filter for only significantly expressed genes/transcripts (using the Filtering tool).

Finally, these questions are best asked on the galaxy-user mailing list, where you can communicate with the Galaxy user community: http://user.list.galaxyproject.org/

Good luck,
J.
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Old 11-28-2012, 03:24 PM   #25
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@Peppe re: JGI gene annotation

No need to run that workflow on the annotation in your case. Just make sure that your gene annotation's contig names (column 1) match the names in your genome's FASTA file.

J.
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Old 11-28-2012, 03:36 PM   #26
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Thanks Jeremy.
I will use the Galaxy user community: http://user.list.galaxyproject.org/
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Old 12-06-2014, 01:16 PM   #27
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I posted a partial solution on iplant's community forum to concerning on how to view Cuffdiff output from Galaxy to cummerbund: http://ask.iplantcollaborative.org/q...-gff3gtf-file/

I hope this helps everyone in the future.
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Old 11-07-2016, 01:02 AM   #28
suparmin
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Hello everyone, my name is andrean, i'm starting to do analysis my RNA sequencing, i want to make a heatmap, and GO analysis, could you tell me how to do using R or another open source program, thank you
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