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Old 02-21-2011, 06:44 PM   #1
kouroshz
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Location: Boston

Join Date: Jul 2010
Posts: 4
Default RNA Seq Poly A, TopHat

Hi Everybody,

I have fastq files of PolyA+ paired-end RNASeq and I need to obtain the fasta or bed files of the actual transcripts, their coding regions, and their UTRs. I have a few questions and I wonder if anybody can kindly help me.

1) Do I need to trim the polyA before the alignment? If so what is the best way to perform this?

2) TopHat combined with cufflinks seem to be able to give you the splice junctions as well as the assembly of the transcripts. Can I also obtain the bed files of the coding regions and the UTRs using this method?

3) Tophat (Bowtie) need the inner distance between paired reads as a parameter. Is the inner distance roughly the average fragment size minus 2 times the read length? Aso I do not have the average lenght, all I know is that the RNAs ar > 200 nt. So what is the best parameter to use?

Thank you,

kz
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