Hi,

I have done exactly the same kind of thing for adenovirus so I can help with it. In answer to question 1 you do not need to index it will be done for you when tophat is called. Secondly you should leave the 40 multihits as it is and post analysis filter out the multihits - this will allow you to determine if you do have a multihit problem or not and if so whether it is a big problem and where it is on the genome. I have a workflow on Galaxy which you can use called "Bristol workflow to get sorted unique proper pair mapped reads". If you plug in your sam file it should give you files listing only unique hits and those which map more than once. This workflow assumes you have paired end data but it can be modified to work with single end reads as well.

Hope this helps.


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

On 6 May 2011, at 17:09, puvan001@umn.edu wrote:

Hi

I have a couple of questions regarding RNA seq analysis. My questions are
1.I need to use a viral genome (very small, ~2kb ) as a reference genome and it is not available in Galaxy. I guess I can use this data from my history. I have a fasta file but I am not sure whether I have to do some kind of indexing or not.

2. In Tophat, default for "maximum number of alignments to be allowed" is 40. What my understanding is a single read can be aligned maximum 40 different places. I am wondering why this is 40. Is there any specific reason? If I need unique mapping, I have to use 1 instead of 40. Am I correct?


Thanks

SP



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