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Old 02-06-2012, 08:39 AM   #1
guzhi100
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Location: germany

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Default RNA-Seq analysis using galaxy

I am a Galaxy user. At this moment, I am using tophat and cufflinks to analyze my RNA-Seq data. I have some questions concerning these tools, and hope you can help me to figure out how to get proper output data.



At the end of the analysis, I expect two lists: differentially expressed transcripts and differentially expressed genes. In these two lists, I would like to see the gene name, gene ID and transcript ID.




What I did is:

After mapping with tophat, I run cufflinks using reference gene sets (GTF file) from Ensembl. I modified the ensembl GTF file according to http://main.g2.bx.psu.edu/u/jeremy/p...lysis-faq#faq4 so that cufflinks can recognize the column for chromosomes. I got the file "cufflinks assembled transcript" which shows nicely the gene ID, transcript ID , but the gene name was lost in this file.



Then I run cuffcompare using the same reference gene sets (GTF file) from Ensembl. In the output file I can see that gene name appeared, but galaxy assigned new ID to gene and transcript.



Then I run cuffdiff . Output file only contains gene name.



My question is: how can I keep the information (gene ID, transcript ID, gene name ) from the reference gene sets during the whole analysis process so that I get meaningful information. Or is that possible that I retrieve "gene ID, transcripte ID, gene name" by using the output file "Cuffdiff transcript differential expression! " from cuffdiff?

I hope you can help me.

Thanks in advance.
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Old 02-06-2012, 01:15 PM   #2
sheenams
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Default

I solved this issue by following the tutorial at the end of this web page:

https://docs.uabgrid.uab.edu/wiki/UA...n_using_TopHat

You'll have to figure out which columns you actually want and specify those in the 'cut' step.
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Old 02-07-2012, 03:05 AM   #3
guzhi100
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Default Thanks

Hi sheenams,

Thanks so much. Actually, I read this tutorial days ago, but didn't pay attention to the last part. Now I got it.
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Old 07-17-2012, 12:39 AM   #4
SilviaBCE
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Question Nucleotide distribution chart

Hi, I'm analyzing my small-RNA-seq data (Illumina 1.9 quality score) and I'm using galaxy to make the preliminary qc tasks. I find it a great and easy tool! I'm here to ask you how can I interpretate a graph:I'm talking about the nucleotide distribution chart after the sample grooming and the 3' adapter trimming. I attach it here so anybody can see it. Up to now I've loaded two samples in galaxy and they both give me this kind of bias at the 3rd nucleotide of the reads. What does it mean? would you suggest to eliminate all those reads which contain the "N" in the 3rd position?
Any suggestion would be appreciated! Thanks a lot.
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