Hi,

Tophat may still be an option for you. You can filter out spliced reads by 
filtering column 6 (the CIGAR column) for reads that only map directly (i.e. 
c6=='56M' if you have a 56bp paired end read). But I agree with Jen that most 
likely it is a sort problem.


Best Wishes,
David.

__________________________________
Dr David A. Matthews

Senior Lecturer in Virology
Room E49
Department of Cellular and Molecular Medicine,
School of Medical Sciences
University Walk,
University of Bristol
Bristol.
BS8 1TD
U.K.

Tel. +44 117 3312058
Fax. +44 117 3312091

d.a.matth...@bristol.ac.uk






On 9 Aug 2011, at 07:27, yao chen wrote:

> Hi Mete,
> 
> I am not sure it is the "sort" problem. I find "cufflinks" in galaxy is 
> unstable. I have bam files from Tophat which I can run cufflinks a few days 
> agao. 
> 
> But these days when I run cufflinks with these bam files, the error shows. 
> Strangely, it can work some time. I don't know the reason.
> 
> ChenYao
> 
> 2011/8/9 Jennifer Jackson <j...@bx.psu.edu>
> Hi Mete,
> 
> This FAQ has a workflow for sorting a Bowtie (or any) SAM file for Cufflinks:
> http://main.g2.bx.psu.edu/u/jeremy/p/transcriptome-analysis-faq#faq2
> 
> Thanks!
> 
> Jen
> Galaxy team
> 
> 
> On 8/4/11 10:27 AM, Mete Civelek wrote:
> Hi,
> 
> I'm trying to get read counts or FPKM values for my miRNA NGS data on
> Galaxy. I have aligned the reads using Bowtie, but it appears that
> Cufflinks gives an error when run on the Bowtie alignments (This might
> have something to do with Bowtie's BAM file not being sorted). I know
> that Tophat alignments work well with Cufflinks, but I'm not sure if it
> would be possible to use Tophat for my data since miRNA don't have
> splice junctions. I've tried without success to parameterize Tophat to
> completely avoid assigning splice junctions (by setting the max intron
> length to 1). Is there a way I can get the Bowtie alignment to work with
> Cufflinks on Galaxy? Or perhaps there's a way I can parametrize Tophat
> as to get no splice junctions?
> 
> Thanks,
> 
> Mete
> 
> 
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