Thank you very much for your reply.
I have one more question about the "Anchor length". For a RNA-seq read mapped
on the splicing junction under the 0 mismatch condition, if 5 nucleotides of
one end map on one exon, does it mean the rest part of the read must map on the
adjacent exon? What I want to understand is that, although reducing "Anchor
length" may reduce the reliability of mapping on one end/exon, but the
increased number of mapped nucleotides on the adjacent exon may increase the
reliability of mapping. Does it mean overall the reliability of mapping is not
From: Jeremy Goecks [jeremy.goe...@emory.edu]
Sent: Wednesday, April 10, 2013 3:16 PM
To: Du, Jianguang
Subject: Re: [galaxy-user] Are reads of 36nt in length long enough to accutatly
map on splicing junctions?
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.
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