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.
Development
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.
Principles
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.Base identification
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.Types
Biological
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
Alpha hemolysin, 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, and is able to discriminate between each base.Sequencing using αHL has been developed through basic study and structural mutations, moving towards the sequencing of very long reads. Protein mutation of αHL has improved the detection abilities of the pore. The next proposed step is to bind an exonuclease onto the αHL pore. The enzyme would periodically cleave single bases, enabling the pore to identify successive bases. Coupling an exonuclease to the biological pore would slow the translocation of the DNA through the pore, and increase the accuracy of data acquisition.
Notably, theorists have shown that sequencing via exonuclease enzymes as described here is not feasible. This is mainly due to diffusion related effects imposing a limit on the capture probability of each nucleotide as it is cleaved. This results in a significant probability that a nucleotide is either not captured before it diffuses into the bulk or captured out of order, and therefore is not properly sequenced by the nanopore, leading to insertion and deletion errors. Therefore, major changes are needed to this method before it can be considered a viable strategy.
A recent study has pointed to the ability of αHL to detect nucleotides at two separate sites in the lower half of the pore. The R1 and R2 sites enable each base to be monitored twice as it moves through the pore, creating 16 different measurable ionic current values instead of 4. This method improves upon the single read through the nanopore by doubling the sites that the sequence is read per nanopore.
MspA
is the second biological nanopore currently being investigated for DNA sequencing. The MspA pore has been identified as a potential improvement over αHL due to a more favorable structure. The pore is described as a goblet with a thick rim and a diameter of 1.2 nm at the bottom of the pore. A natural MspA, while favorable for DNA sequencing because of shape and diameter, has a negative core that prohibited single stranded DNA translocation. The natural nanopore was modified to improve translocation by replacing three negatively charged aspartic acids with neutral asparagines.The electric current detection of nucleotides across the membrane has been shown to be tenfold more specific than αHL for identifying bases. Utilizing this improved specificity, a group at the University of Washington has proposed using double stranded DNA between each single stranded molecule to hold the base in the reading section of the pore. The dsDNA would halt the base in the correct section of the pore and enable identification of the nucleotide. A 2011 grant was awarded to a collaboration from UC Santa Cruz, the University of Washington, and Northeastern University to improve the base recognition of MspA using phi29 polymerase in conjunction with the pore. MspA with electric current detection can also be used to sequence peptides.
CsgG
The CsgG nanopore is a 36-stranded β-barrel protein from Escherichia coli. The CsgG pore has a nine-fold circular symmetry and a single, well-defined constriction that is approximately 1 nm wide.A significant development in its use for sequencing is the creation of a dual-constriction pore by combining CsgG with its partner protein, CsgF. The N-terminal region of CsgF binds inside the CsgG barrel, creating a second constriction that is roughly 1.5 nm wide and separated from the original CsgG constriction by about 2.5 nm. This dual-constriction structure is a major advantage because both constrictions contribute to the electrical signal modulation as a single strand of DNA moves through the pore.
This dual-reading capability significantly improves sequencing accuracy, particularly for homopolymer regions, which are a known source of errors in nanopore sequencing. DNA sequencing using this dual-constriction CsgG:CsgF pore has been shown to improve single-read accuracy by 25-70% in homopolymers up to 9 nucleotides long. The addition of the second constriction increases the signal complexity, leading to higher accuracy in calling the length of homopolymers compared to a single-constriction CsgG pore.
Solid state
Solid state nanopore sequencing approaches, unlike biological nanopore sequencing, do not incorporate proteins into their systems. Instead, solid state nanopore technology uses various metal or metal alloy substrates with nanometer sized pores that allow DNA or RNA to pass through. These substrates most often serve integral roles in the sequence recognition of nucleic acids as they translocate through the channels along the substrates.Tunneling current
Measurement of electron tunneling through bases as ssDNA translocates through the nanopore is an improved solid state nanopore sequencing method. Most research has focused on proving bases could be determined using electron tunneling. These studies were conducted using a scanning probe microscope as the sensing electrode, and have proved that bases can be identified by specific tunneling currents. After the proof of principle research, a functional system must be created to couple the solid state pore and sensing devices.Researchers at the Harvard Nanopore group have engineered solid state pores with single walled carbon nanotubes across the diameter of the pore. Arrays of pores are created and chemical vapor deposition is used to create nanotubes that grow across the array. Once a nanotube has grown across a pore, the diameter of the pore is adjusted to the desired size. Successful creation of a nanotube coupled with a pore is an important step towards identifying bases as the ssDNA translocates through the solid state pore.
Another method is the use of nanoelectrodes on either side of a pore. The electrodes are specifically created to enable a solid state nanopore's formation between the two electrodes. This technology could be used to not only sense the bases but to help control base translocation speed and orientation.
Another technique to support ionic current based sensing is called Nanopore electrometry, which has been also recently proposed theoretically. This is where the modulations in electric field is utilised instead of the modulations in ionic current for nanopore sensing.