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 sensingIonic current passed through membrane by setting a voltage across the membrane.Disruption in current detected when analyte passes through the poreor near its aperture.Characteristic disruption indentifies the molecule in question.
4 Nanopore DNA sequencing DNA polymer or individual nucleotides pass through the nanopore.Detected bya adaptor molecule ( e.g. Cyclodextrin).Tunnelling electrodes based detectors.Capacitive detectorsGraphene 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 processive exonuclease.
7 Electron-based read out Four different magnitudes of disruption which can be classified as C, G, A or TModified 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 AnalysisSnapshot from movie at
10 Exonuclease Sequencing Adapter molecule (cyclodextrin):Accuracy averaging 99.8%Identification of meCSnapshot from movie at
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: NodeCan be used in a cluster, connected through network.Low power and space required.Permits scheduling and multiplexing.
13 Workflow versatility No fixed run time Run until... sufficient data 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 dataThe GridION system enables users to run an experiment until sufficient data has been collected to reach a predetermined experimental endpoint.
15 Oxford nanoporeDNA sequencing: applications BesidesPersonalised MedicineDiagnosis and treatmentPharmacogenomicsPreventionSecurity/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.