> 1) My reads are 36nt long. How much should I set for "the Minimum length of 
> reads segments" to get the most reliable output with the highest mapping of 
> splicing junctions?. In my previous run of TopHat, I set it as 18. Can I 
> reduce it more to get better mapping on splicing junctions?

You'll need to define for yourself what you mean by "better/best mapping" and 
experiment to find the parameters that give you the best results.

> 2) I do not understand exactly how TopHat works as for the "Anchor length" 
> although I have read the manual for TopHat. 
> Suppose I set the "Anchor length as 8 and the "Maximum number of mismatch 
> that can appear in the anchor region of spliced alignment" as 0 when I run 
> Tophat. Does it mean, for a read maps on two adjacent exons, TopHat will 
> report this alignment to the outputs ".accepted hits" and ".splicing 
> junctions" if either end of the read has 8 or more nucleotides mapping on one 
> exon?

I think that's correct.

> 3) Is there disadvantage/negative effect if I choose to set the "Anchor 
> length" at the lowest, for example 3? My understanding is that, under the 0 
> mismatch condition, if 3 nuceoides of one end of a read mapped on one exon, 
> the other part of the read will map on the adjacent exon (in my case, the 
> other part would be 33 nucleotides). So my understanding is that setting the 
> "Anchor length" at 3 does not increase the inaccuracy of the alignment. Am I 
> correct?

Setting the anchor length especially small reduces the constraints on mapping, 
so more reads will map but there are likely to be more false positives as well.

Good luck,
J.
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