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
Nanopore sensing Disruption in current detected when analyte passes through the pore or near its aperture. Characteristic disruption indentifies the molecule in question. Ionic current passed through membrane by setting a voltage across the membrane.
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 Snapshot from movie at http://www.nanoporetech.com Hairpin structure: Sense and anti-sense sequencing Advantages in Data Analysis
Exonuclease Sequencing Snapshot from movie at http://www.nanoporetech.com
Exonuclease Sequencing Snapshot from movie at http://www.nanoporetech.com Adapter molecule (cyclodextrin): Accuracy averaging 99.8% Identification of meC
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.