Oxford Nanopore Technologies Nanopore Sequencing Introduction to nanopore

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 me. C Snapshot from movie at http: //www. nanoporetech. com

Working strategy • Min. ION: a miniaturised sensing instrument – Portable. – Field-deployable. – Requires minimal sample prep. – Compatible with blood serum, plasma and whole blood.

Working strategy • Grid. ION 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 Grid. ION system enables users to run an experiment until sufficient data has been collected to reach a predetermined experimental endpoint.

Run until. . . sufficient data

Oxford nanopore. DNA 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.

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