Oxford Nanopore Technologies

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Oxford Nanopore Technologies
Nanopore Sequencing
Introduction to nanopore sensing
A nanopore: a nano-scale hole.
•
Biological: a pore-forming protein (e.g. αHemolysin) in a membrane (e.g. lipid bilayer)
•
Solid-state: in synthetic materials ( e.g. silicon
nitride or graphene)
•
Hybrid: formed by a pore-forming protein set in
synthetic material
Nanopore sensing
Ionic current passed through membrane by setting a voltage
across the membrane.
•Disruption in current detected when analyte passes through the pore
or near its aperture.
•Characteristic disruption indentifies the molecule in question.
Nanopore DNA sequencing
• DNA polymer or individual nucleotides
pass through the nanopore.
• Detected by
– a adaptor molecule ( e.g. Cyclodextrin).
– Tunnelling electrodes based detectors.
– Capacitive detectors
– Graphene based nano-gap or edge state
detectors.
Nanopore DNA sequencing
• Strand sequencing:
– Sequencing in real-time as the intact DNA
polymer passes through the nanopore.
• Exonuclease sequencing:
– Individual nucleotides pass through the
nanopore by the aid of processive
exonuclease.
Strand Sequencing
Snapshot from movie at http://www.nanoporetech.com
Electron-based read out
Four different magnitudes of
disruption which can be classified as
C, G, A or T
Modified base, e.g. methylated
cytosine, can be directly
distinguished from the four standard
bases
Strand Sequencing
 Hairpin structure:
Sense and anti-sense sequencing
Advantages in Data Analysis
Snapshot from movie at http://www.nanoporetech.com
Exonuclease Sequencing
Snapshot from movie at http://www.nanoporetech.com
Exonuclease Sequencing
 Adapter molecule (cyclodextrin):
• Accuracy averaging 99.8%
• Identification of meC
Snapshot from movie at http://www.nanoporetech.com
Working strategy
• MinION: a miniaturised sensing instrument
– Portable.
– Field-deployable.
– Requires minimal sample prep.
– Compatible with blood serum,
plasma and whole blood.
Working strategy
• GridION system
– Uses single-use, self-contained
cartridge.
– Can be used as a single instrument:
Node
– Can be used in a cluster, connected
through network.
– Low power and space required.
– Permits scheduling and multiplexing.
Workflow versatility
• No fixed run time
– Can be run one or more nodes for minutes or days.
– Data analysis takes place in real time.
– Longer run enables collecting more data points.
• Run until... sufficient data
– The GridION system enables users to run an experiment until
sufficient data has been collected to reach a predetermined
experimental endpoint.
Run until... sufficient data
Oxford nanoporeDNA sequencing:
applications
Besides
–
–
–
–
–
Personalised Medicine
Diagnosis and treatment
Pharmacogenomics
Prevention
Security/defence
Advantages over present sequencing
technologies
•
•
•
•
•
•
•
Real-time sequencing strategy.
No strand amplification needed.
No bias due to sequencing amplification.
Low cost: trying to fulfil the target of $1000 per human genome.
Lager read size: read size is limited only by preparation.
No requirement for large amounts of high-performance disk storage.
Large-scale structural variation can be detected at lower depth of
coverage.
• Enable long-range haplotyping.
• No need for expensive and time-consuming mate pair library
construction.
Thank you
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