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Old 12-09-2010, 12:06 PM   #2
Thomas Doktor
Senior Member
Location: University of Southern Denmark (SDU), Denmark

Join Date: Apr 2009
Posts: 105

First of all, I'm not sure if you have used Bowtie itself or TopHat, which uses Bowtie as it's aligner?

In any case, I don't think you can completely eliminate the problem with misaligning reads right at the intron-exon border, because you need a certain minimum length of coverage on either side of the intron in order to map a read across an exon junction. What I would do, is filter out all variations found within a distance of, say 5 bases, from the exons. This will minimize the number or true positives that you reject, especially considering that this region is already particular sensitive to mutations and seldom have neutral SNPs within them. If you are looking for disease causing mutations which for instance cause intron retention or usage of alternative splice sites, you should be able to detect this and then change the filter for those particular cases.

Another RNA-seq aligner you might try using is MapSplice which, in my experience, does a little better job than TopHat/Bowtie.

Last edited by Thomas Doktor; 12-09-2010 at 12:11 PM.
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