In 2015, Yaniv Erlich outlined a concept of an “Internet of Living Things” made possible by a future of ubiquitous genomics. Erlich noted that, “multiple appliances could benefit from integration with sequencing sensors, including air conditioning or the main water supply to monitor harmful pathogens. However, of all possible options, toilets may offer the best integration point.”
Nanopore sequencing is a scalable technology that enables direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore.
Here are some examples of the state of play in nanopore technology.
DNA Sequencing
The per-base cost of raw DNA sequencing is falling rapidly. Next generation sequencing (NGS) is driven by technologies such as cytosine methylation. Nanopore sequencing is a third generation approach used in the sequencing of biopolymers – specifically, polynucleotides in the form of DNA or RNA.
Nanopores have been studied as a unique DNA sequencing technology that can quickly read long stretched DNA sequences. A DNA molecule could pass through a nanopore in a speed of microsecond per base and even faster. With this speed, a human genome can potentially be sequenced by one nanopore in <1h. In contrast to NGS, the nanopore sequencing is enzyme free without need of sample amplification due to its single-molecule nature. Nanopore sequencing has potential as a new generation of DNA sequencing technology in the post-NGS era.
One challenge for the ‘strand sequencing’ method was in refining the method to improve its resolution to be able to detect single bases. In the early papers methods, a nucleotide needed to be repeated in a sequence about 100 times successively in order to produce a measurable characteristic change. This low resolution is because the DNA strand moves rapidly at the rate of 1 to 5μs per base through the nanopore. This makes recording difficult and prone to background noise, failing in obtaining single-nucleotide resolution. The problem is being tackled by either improving the recording technology or by controlling the speed of DNA strand by various protein engineering strategies and Oxford Nanopore employs a ‘kmer approach’, analyzing more than one base at any one time so that stretches of DNA are subject to repeat interrogation as the strand moves through the nanopore one base at a time.
There is a broader initiative by the Telomere-to-Telomere (T2T) consortium, partially funded by National Human Genome Research Institute (NHGRI), part of the National Institutes of Health (NIH). The consortium aims to generate a complete reference sequence of the human genome.
Researchers recently published a complete sequence of chromosome eight (8)—the first non-sex chromosome to be sequenced completely. The team identified the 2.3% of chromosome 8’s dark genes with long-read instruments from Oxford Nanopore Technologies and Pacific Biosciences. Unlike short-read platforms, the more expensive long-read platforms can sequence ‘hard-to-read’ areas of the genome.
Biological Physiology
Since the discovery of biological ion channels and their role in physiology, scientists have attempted to create man-made structures that mimic their biological counterparts.
New research by Lawrence Livermore National Laboratory (LLNL) scientists and collaborators at the University of California, Irvine shows that synthetic solid-state nanopores can have finely tuned transport behaviors much like the biological channels that allow a neuron to fire.
In biological ion channels, two of the most exciting properties are the ability to respond to external stimuli and differentiate between two ions of the same charge, such as sodium and potassium.
It is well known that synthetic nanopores can distinguish between positive and negative ions (such as potassium and chloride) but in the new research, the team was able to distinguish between sodium and potassium ions despite their equal charge and nearly identical size. The potassium-selective channels showed currents that were roughly 80 times larger for potassium ions than sodium ions, significantly higher than any other man-made system has demonstrated and a first for solid-state nanopores.
“We can use our synthetic platforms to better understand how biological systems work,” said Steven Buchsbaum, LLNL staff scientist and a lead author of a paper appearing in the Feb. 8 edition of Science Advances.
“Performing studies on man-made systems built from the ground up can give unique insight into how these pores function and the underlying physical phenomena behind them.”
UCI professor and collaborator Zuzanna Siwy said the most exciting application for the nanopores is their use as a building block toward making artificial biomimetic systems such as an artificial neuron.
Biology uses ion selectivity to enable energy storage in the form of a chemical potential across a cell membrane. This energy can then be tapped into later, powering processes such as nerve signaling. “The ability to do the same in man-made materials takes us one step closer to making synthetic biomimetic componentry,” Siwy said.
The capability to distinguish between ions that closely resemble each other also can be applied to areas such as desalination/filtration and biosensing.
“Working with synthetic nanopores offers the benefits of increased control over the pore design and using materials that are much more robust than those seen in biology,” said Francesco Fornasiero, LLNL staff scientist and coauthor. “This could enable us to eventually replace or repair biological materials with artificial versions that are superior to their biological counterparts.”
Postdoctoral researcher Elif Turker Acar, graduate student researcher Cody Combs and Istanbul University also contributed to the research.
The work was funded by LLNL’s Laboratory Directed Research and Development program.
