[mira_talk] PacBio CCS questions
- From: "Matthew D. Pagel" <pagel@xxxxxxx>
- To: mira_talk@xxxxxxxxxxxxx
- Date: Wed, 14 Aug 2013 19:34:16 -0400
I was looking at an alignment of filtered but uncorrected reads from PacBio
data (covering a particular area of the genome where PacBio and 454 data were
in apparent disagreement). I noticed that subreads of a given read that was
labelled as "CCS" were not oriented in the same direction. My understanding of
the technology was that a given read tracks a single polymerase in a ZMW. I
also understood that reads are presumed to be of a contiguous sequencing
reaction (no disassociation of polymerase from template). Thus, I would assume
that each subread should be read in the same direction as the previous, given a
small circular piece of DNA resulting from library preparation.
I do note that my subreads alternate in orientation vs the reference along the
CCS read (RFRFRFR). Upon further analysis, approximately 1/4 of my non-CCS
subreads are from reads with multiple subreads (that follow the FR or
RF...never FF or RR). My assumption is that I'm dealing with a region of the
genome that has a inverted duplication. Further the read-to-subread algorithm
is inappropriately identifying adaptor sequence (or I happen to have the
adaptor sequence in my genome verbatim).
Alternatively, does this indicate a problem with the library preparation? Is
the polymerase in the ZMW encountering some secondary structure and jumping to
the other strand of dsDNA? Anything else that you can think of that is going on?
Is there a quick-and-dirty algorithm out there for identifying inversions from
one subread to the next within a single PB read, assuming that these data
really indicate a genomic duplication and inversion rather than a re-read of
the same genomic region? Is there a way to identify "false adaptors"
otherwise, as some genomic duplications are oriented the same direction (FF)
rather than as an inversion?
_______________________________________________________
Matt Pagel
Graduate Student
Penn State Biochemistry, Microbiology and Molecular Biology
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