Noa,
> This is one thing I would like help with- is it worth simply reducing to
> nothing the max intron size? What is accepted consensus when using tophat on
> bacterial genomes?
I'm not sure that folks on this list have much experience with bacterial
transcriptome analysis. You might try seqanswers.com or try emailing the
Tophat/Cufflinks authors directly: tophat.cuffli...@gmail.com If you find
something interesting in another place, please feel free to share with the
Galaxy community.
> When I look at the second tophat file, of accepted hits, all hits align
> nicely with known genes. However, when I run cufflinks I run into the
> following issues: when I use a reference genome, I get in addition to the
> known transcripts, a bunch of very long transcripts spanning very large
> genomic regions. Also, I will have two genes that are very near each other
> but run in opposite directions (which you can see beautifully in the tophat
> accepted hits alignments - different colors for each strand) but they merge
> into a single CUFF identifier. Is there any way I can address this- is it
> something I am missing with respect to parameters I have to change because I
> am working on a bacterial genome?
Reference genome or reference gene annotation? Using a genome to correct for
bias should not change the assembled transcripts, only their expression levels.
You can use a reference gene annotation either as ground truth or as a guide;
using the reference as ground truth ensures that Cufflinks will only assemble
transcripts defined in the annotation.
Good luck,
J.
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