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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