Nanopore sequencing

Nanopore sequencing is a third generation approach used in the sequencing of biopolymers — specifically, polynucleotides in the form of DNA or RNA.

Nanopore sequencing allows a single molecule of DNA or RNA be sequenced without PCR amplification or chemical labeling. Nanopore sequencing has the potential to offer relatively low-cost genotyping, high mobility for testing, and rapid processing of samples, including the ability to display real-time results. It has been proposed for rapid identification of viral pathogens, monitoring ebola, environmental monitoring, food safety monitoring, human genome sequencing, plant genome sequencing, monitoring of antibiotic resistance, haplotyping and other applications.

Nanopore sequencing took 25 years to materialize. David Deamer was one of the first to push the idea. In 1989 he sketched out a plan to push single-strands of DNA through a protein nanopore embedded into a thin membrane as part his work to synthesize RNA. Realizing that the approach might allow DNA sequencing, Deamer and his team spent a decade refining the concept. In 1999 they published the first paper using the term 'nanopore sequencing' and two years later produced an image capturing a DNA hairpin passing through a nanopore in real time.

Another foundation for nanopore sequencing was the work of Hagan Bayley's team, who from the 1990s independently developed stochastic sensing, a technique that measures the change in an ionic current passing through a nanopore to determine the concentration and identity of a substance. By 2005 Bayley had made progress with the DNA sequencing method. He co-founded Oxford Nanopore to push the technology. In 2014 the company released its first portable nanopore sequencing device. This made it possible for DNA sequencing to be carried out almost anywhere, even with limited resources. A quarter of the world's SARS-CoV-2 viral genomes were sequenced with nanopore devices. The technology offers an important tool for combating antimicrobial resistance.

The biological or solid-state membrane, where the nanopore is found, is surrounded by an electrolyte solution. The membrane splits the solution into two chambers. Applying a bias voltage across the membrane induces an electric field that drives charged particles, in this case the ions, into motion. This effect is known as electrophoresis. For high enough concentrations, the electrolyte solution is well distributed and the voltage drop concentrates near and inside the nanopore. This means charged particles in the solution feel a force only from the electric field when they are near the pore region. This region is typically referred to as the capture region. Inside the capture region, ions have a directed motion that can be recorded as a steady ionic current by placing electrodes near the membrane. A nano-sized polymer such as DNA or protein placed in one of the chambers has a net charge that feels a force from the electric field in the capture region. The molecule approaches this capture region aided by Brownian motion. Any attraction it might have to the surface of the membrane. Once inside the nanopore, the molecule translocates via a combination of electro-phoretic, electro-osmotic and sometimes thermo-phoretic forces. Inside the pore the molecule occupies a volume that partially restricts the ion flow, observed as an ionic current drop. Based on various factors such as geometry, size and chemical composition, the change in magnitude of the ionic current and the duration of the translocation vary. Different molecules can then be sensed and potentially identified based on this current modulation.

The magnitude of the electric current density across a nanopore surface depends on the nanopore's dimensions and the composition of DNA or RNA that is occupying the nanopore. Sequencing was made possible because passing through the channel of the nanopore, the samples cause characteristic changes in the density of the electric current. The total charge flowing through a nanopore channel is equal to the surface integral of electric current density flux across the nanopore unit normal surfaces.

Biological nanopore sequencing relies on the use of transmembrane proteins, called protein nanopores, in particular, formed by protein toxins, that are embedded in lipid membranes so as to create size dependent porous surfaces - with nanometer scale "holes" distributed across the membranes. Sufficiently low translocation velocity can be attained through the incorporation of various proteins that facilitate the movement of DNA or RNA through the pores of the lipid membranes.

Alpha hemolysin (αHL), a nanopore from bacteria that causes lysis of red blood cells, has been studied for over 15 years. To this point, studies have shown that all four bases can be identified using ionic current measured across the αHL pore. The structure of αHL is advantageous to identify specific bases moving through the pore. The αHL pore is ~10 nm long, with two distinct 5 nm sections. The upper section consists of a larger, vestibule-like structure and the lower section consists of three possible recognition sites (R1, R2, R3), and is able to discriminate between each base.