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.Matthews@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 <jen@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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