Re: [galaxy-user] [galaxy-bugs] Cufflinks
Hi Gabor, I'm moving your email to the galaxy-user mailing list because it concerns galaxy usage; also, there's a substantial community of users doing RNA-seq that may be able to offer suggestions to help you out. To your issue:
I used Galaxy (Bowtie) to successfully map 15 million Illumina reads trimmed to 65bp. When I applied Cufflinks to the BAM data no transcripts were reported even though it ran OK. It is possible that very little mapped to known exons in this data set. Does Cufflinks only report data for known transcripts? I thought it was designed to work without a reference annotation. How does it decide what qualifies as a transcript? Any ideas why I got such a result?
Your problems are likely at least partially due to using Bowtie instead of Tophat. Tophat is the standard way to map reads so that Cufflinks can assemble transcripts. Cufflinks assembles transcript--de novo or reference-guided--based on the mapped reads, but mapped reads must include spliced reads, which are generated by Tophat but not Bowtie. Good luck, J.
Hi, I agree with Jeremy on this one, you should use tophat as that is what cufflinks is expecting - particularly the fact that the samfile is sorted correctly which I'm not certain bowtie does. The other thing that may be wrong is that your chromosome annotation may not match if you used a different reference genome for the alignment compared to the reference genome you told cufflinks to use (I did that once!). As Jeremy says cufflinks should be OK without a gene annotation file. Cheers David On 7 Apr 2011, at 16:11, Jeremy Goecks wrote:
Hi Gabor,
I'm moving your email to the galaxy-user mailing list because it concerns galaxy usage; also, there's a substantial community of users doing RNA-seq that may be able to offer suggestions to help you out.
To your issue:
I used Galaxy (Bowtie) to successfully map 15 million Illumina reads trimmed to 65bp. When I applied Cufflinks to the BAM data no transcripts were reported even though it ran OK. It is possible that very little mapped to known exons in this data set. Does Cufflinks only report data for known transcripts? I thought it was designed to work without a reference annotation. How does it decide what qualifies as a transcript? Any ideas why I got such a result?
Your problems are likely at least partially due to using Bowtie instead of Tophat. Tophat is the standard way to map reads so that Cufflinks can assemble transcripts. Cufflinks assembles transcript--de novo or reference-guided--based on the mapped reads, but mapped reads must include spliced reads, which are generated by Tophat but not Bowtie.
Good luck, J.
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I think if Tophat is used it will call Bowtie. Vasu --- On Thu, 4/7/11, Jeremy Goecks <jeremy.goecks@emory.edu> wrote: From: Jeremy Goecks <jeremy.goecks@emory.edu> Subject: Re: [galaxy-user] [galaxy-bugs] Cufflinks To: "Gabor Bartha" <gabor@synergenics.net> Cc: "galaxy-user" <galaxy-user@lists.bx.psu.edu> Date: Thursday, April 7, 2011, 10:11 AM Hi Gabor, I'm moving your email to the galaxy-user mailing list because it concerns galaxy usage; also, there's a substantial community of users doing RNA-seq that may be able to offer suggestions to help you out. To your issue: I used Galaxy (Bowtie) to successfully map 15 million Illumina reads trimmed to 65bp. When I applied Cufflinks to the BAM data no transcripts were reported even though it ran OK. It is possible that very little mapped to known exons in this data set. Does Cufflinks only report data for known transcripts? I thought it was designed to work without a reference annotation. How does it decide what qualifies as a transcript? Any ideas why I got such a result? Your problems are likely at least partially due to using Bowtie instead of Tophat. Tophat is the standard way to map reads so that Cufflinks can assemble transcripts. Cufflinks assembles transcript--de novo or reference-guided--based on the mapped reads, but mapped reads must include spliced reads, which are generated by Tophat but not Bowtie. Good luck, J. -----Inline Attachment Follows----- ___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/
participants (3)
-
David Matthews -
Jeremy Goecks -
vasu punj