Question: Can Tophat Deal With Overlapping Transcripts When Given --Gtf ?
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gravatar for bw.
5.5 years ago by
bw.150
San Francisco
bw.150 wrote:

I'd like to run TopHat2 on a 50bp single-end rna-seq dataset in order to get gene counts for differential expression analysis. I was going to run with --GTF ensembl_genes.gtf since the TopHat2 paper talks about how this leads to significant gains in sensitivity and accuracy. What I'm wondering is - how will overlapping ensembl transcripts effect the results?
When TopHat generates a fasta from my ensembl gene file, it includes multiple overlapping sequences. It seems like these would lead to ambiguous alignment, and that I need to merge overlaps before running TopHat, but I'm not finding any discussion of this on the forum or in the papers, so wanted to double check.

Thanks -Ben

gtf tophat • 1.7k views
ADD COMMENTlink modified 5.5 years ago • written 5.5 years ago by bw.150
0
gravatar for Sean Davis
5.5 years ago by
Sean Davis25k
National Institutes of Health, Bethesda, MD
Sean Davis25k wrote:

TopHat deals with this situation just fine. In fact, at the mapping stage, overlapping transcripts are not really a big problem. However, such overlapping transcripts are harder to deal with at the quantification step (after alignment). This is what cufflinks and other quantification softwares try to deal with.

ADD COMMENTlink written 5.5 years ago by Sean Davis25k

That is not entirely true. Tophat may mispredict novel splice junctions by choosing a splicing motif from the wrong strand.

ADD REPLYlink written 5.5 years ago by Gww2.6k

TopHat is certainly designed with the problem of overlapping transcripts as well as antisense transcripts in mind, but you are certainly correct that TopHat can and does produce false positive (and false negative) results.

ADD REPLYlink written 5.5 years ago by Sean Davis25k
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