[mira_talk] Presenting PCR data as paired end reads?
- From: Peter <peter@xxxxxxxxxxxxxxxxxxxxx>
- To: mira_talk@xxxxxxxxxxxxx
- Date: Mon, 28 Jun 2010 12:01:15 +0100
Hi Bastien et al.,
A common task in finishing is gap closing - where you try to join
together contigs by PCR and Sanger sequencing (e.g. when based on a
reference genome or other evidence you have reason to believe two
contigs should be joined). You would then design primers intended to
be specific to these contig ends (based on what is known of the
genome), try and do the PCR, measure the product size on a gel, and
maybe extract the band and Sanger capillary sequence it.
If we do all that, and get a nice sequence for the whole PCR fragment
I can then do a new assembly incorporating this new Sanger read - and
hopefully this will stitch the two contigs together. Or do this
manually in an editor.
What if the PCR fragment was too big to Sanger sequence in one go, but
we have partial sequence from either end? This could be presented as
paired end reads (of length say 400bp each with about 200bp missing
between them for an expected total length of 1000bp).
However, suppose we don't do the Sanger sequencing. What if we have
just the fact that a pair of PCR primers amplified a region of about
(say) 1000bp? This could be presented as a paired end read (the
forward and reverse primer sequences of say 20bp each). Do you think
it would be useful to try and present this information to MIRA or are
such "reads" just too small?
What do you think? Have you (or anyone else) tried this sort of thing
with MIRA? Would I be better off trying a dedicated scaffolding
program?
Thanks,
Peter
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