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Abstract
The MinIONTM is a miniature nanopore-based analysis device in which the characteristics of an analyte, as it passes through the nanopore, cause changes in the flow of ions through the pore, which are measured, as current flow, by a low noise amplifier and analogue-to-digital converter. Potentially any molecular analyte capable of passing through the nanopore may modify the flow of ions and generate a signal which might be diagnostic. In practice the current device is focussed on DNA sequencing, directly sequencing RNA is a likely development. With the MinION Access Program making the MinION TM widely available a flood of applications exploiting its real time, long read capabilities have been published. We review the background to the technology and compare it to current next generation sequencing.
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Figure 2.
Current flow through a-hemolysin (Reproduced with Permission from Cherf et al., 2012 (66))
Figure 3.
Blocking oligomer for phi29 DNA polymerase motor (Reproduced with Permission from Cherf et al., 2012 (66)).
Figure 4.
(A) MinION MkI (B) flow cell (C) nanopore array (Individual nanopore cells reproduced, modified, with permission from Oxford Nanopore).
Figure 5.
Read accuracy over the start of the MinION Access Program 2014 (Reproduced with Permission, from Loman and Watson, 2015 (46)).
Figure 6.
Example protocol for SQK-MAP006 (September 2015). FFPE – Formalin Fixed Paraffin Embedded DNA repair kit. One step end repair/dA tail NEB Ultra II kit. Magnetic beads -Agencourt AMPure XP beads. MyOne Streptavidin magnetic beads.
Figure 7.
Molecular events and ionic-current trace for a 2D read of an M13 phage dsDNA molecule. (A) Steps in DNA translocation through the nanopore: (i) open channel; (ii) dsDNA with lead adaptor (blue), bound molecular motor (orange) and hairpin adaptor (red) is captured by the nanopore; capture is followed by translocation of the (iii) lead adaptor, (iv) template strand (gold), (v) hairpin adaptor, (vi) complement strand (dark blue) and (vii) trailing adaptor (brown); and (viii) status returns to open channel. (B) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the trace corresponding to steps i-viii are labeled. (C) Expanded time and current scale for raw current traces corresponding to steps i-viii. Each adaptor generates a unique current signal used to aid base calling. (Reproduced with Permission, from Jain et al., 2015 (89))
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