Oxford Nanopore Technologies

1. Oxford Nanopore Technologies Nanopore Sequencing

2. Introduction to nanopore sensing A nanopore: a nano-scale hole. • Biological: a pore-forming protein (e.g. α-Hemolysin) 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

3. 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.

4. 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.

5. 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 processiveexonuclease.

6. Strand Sequencing Snapshot from movie at http://www.nanoporetech.com

7. 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

8. Strand Sequencing • Hairpin structure: • Sense and anti-sense sequencing • Advantages in Data Analysis Snapshot from movie at http://www.nanoporetech.com

9. Exonuclease Sequencing Snapshot from movie at http://www.nanoporetech.com

10. Exonuclease Sequencing • Adapter molecule (cyclodextrin): • Accuracy averaging 99.8% • Identification of meC Snapshot from movie at http://www.nanoporetech.com

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

12. 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.

13. 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.

14. Run until... sufficient data

15. Oxford nanoporeDNA sequencing: applications Besides • Personalised Medicine • Diagnosis and treatment • Pharmacogenomics • Prevention • Security/defence

16. 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.