> 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
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
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.
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