Nanopore-based methods took over 25 years to fully materialise, which involved the collaboration of both academia and industry. It is a third-generation NGS approach that is believed to be one of the most promising sequencing technologies and is hoped to drive genomic research forwards in the near future. Nanopore sequencing has the potential to offer cost-effective genotyping, high mobility for testing and rapid real-time processing of samples.
The advent of nanopore sequencing
The concept of nanopore sequencing was first hatched in 1989 by a research professor of biomolecular engineering, called David Deamer. He explored what would happen if a DNA strand passed through a membrane channel under voltage. Six years later, Deamer and other professors developed the idea into an application for the US Patent Office, who apparently found the concept ‘wild’. Nevertheless, in 1999 the team published their first paper using the term ‘nanopore sequencing’, and two years later produced an image that captured a hairpin of DNA passing through a nanopore. This was a totally novel way of sequencing DNA.
Meanwhile, professor Hagan Bayley and his team at the University of Oxford were independently working on a technique that measured the changes in ionic current passing through a nanopore to identify a substance. Bayley made significant progress on the concept, and subsequently co-founded Oxford Nanopore Technologies in 2005. Nine years later, the company released its first portable DNA sequencing machine, called the MinION, which incorporated the nanopore-based method. This was a revelation, as it allowed DNA sequencing to be carried out almost anywhere. In 2014, the MinION was capable of threading 30 base pairs per second. Since then, the translocation of DNA through nanopores has increased by over 15-fold, which was also accompanied by an increase in read quality.
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The prospects of nanopore sequencing
Currently, the platforms of Oxford Nanopore Technologies include the pocket-sized MinION, the larger GridION and the bench top PromethION. In 2018, it was reported that the MinION could be used to sequence an entire human genome and compare it to a reference. As the technology doesn’t use PCR amplification, the epigenetic modifications were maintained and were actually measurable. Also, the mapping of several ultra-long reads with telomeric repeats to specific chromosomal regions was possible. Essentially, this handheld MinION device was proven to be capable of obtaining reads of a complex genome, up to 882 kilobases in length. This accomplishment indicated that the future of nanopore sequencing was hugely exciting.
Astonishingly, the MinION only costs around $1,000 and is just a fraction larger than the average USB drive. Its extreme portability is perhaps its major advantage. There is no need for complex library preparation and RNA sequencing using the MinION doesn’t require the conversion to complementary DNA first.
It is almost certain that the future of DNA sequencing will consist of machines that are able to be used directly in the field – but there are obstacles to overcome first. Today, the main issue is that most molecular methods are restricted to being carried out in clean and well-equipped facilities. Most scientists are accustomed to in-laboratory analysis, so the lack of portability in typical NGS machines is not a significant problem for a lot of projects. This means that researchers are not turning towards handheld platforms, potentially slowing their success rate.
A photograph of the MinION nanopore machine, demonstrating its small size. Image credit: E. Cairns, 2018
Nevertheless, the COVID pandemic may be driving the desire for portable NGS sequencing. Recently, the Oxford Nanopore Technologies announced the rollout of LamPORE – a COVID test that is designed to work with swabs and saliva samples. The platform uses a combination of loop-mediated isothermal amplification and nanopore sequencing. The technology is not only capable of identifying three genes of the SARS-CoV-2 infection, but it can also be used to test for other pathogens in a single sample, such as respiratory syncytial virus. A pilot study at the beginning of 2021 found that the LamPORE demonstrated an accuracy of over 99.5%, in both symptomatic and asymptomatic individuals. The release of the machine is particularly exciting because it is scalable and able to rapid provide screening in public spaces such as airports, nursing homes and schools.
Gordon Sanghera, the CEO of Oxford Nanopore, said: “We designed LamPORE with both centralised and decentralised COVID-19 testing in mind. Deploying the test using mobile laboratories enables the teams to respond to changing testing demands across the UK and deliver thousands of tests per day.”
Since the technology was released, nanopore base calling algorithms have been consistently improved and the pore system lifespans have increased. If the technologies continue to make such progress, they will begin to play an increasing role in clinically significant human sequencing applications. Also, Oxford Nanopore Technologies has been focused on automation solutions that would enable users to run higher numbers of samples, larger projects and free-up more time for other tasks. The company has worked with Hamilton for the automation of a PromethION machine and collaborated with Opentrons to work on automation for the MinION and GridION platforms.
Furthermore, scientists at the Earlham Institute have helped to improve the quality of their biological data by producing a tool called ALVIS, specific to nanopore sequencing, that visualises genome assemblies and gene alignments. Dr Samuel Martin, one of the researchers involved in the project, explained: “Visualisation of alignment data can help us to understand the problem at hand. As a novel technology, several new alignment formats have been implemented by new tools that are specific to nanopore sequencing technology. Alignments are so fundamental to bioinformatics that it could be of use to anyone working with long-read sequencing data and the diagrams that Alvis generates can be easily exported to directly use in publications.”
All of these advances indicate that nanopore approaches could lead the way in achieving the ‘everyday routine full genome workup’ that scientists are striving for. The future of nanopore sequencing is most certainly bright with the advent of such advanced supporting technologies and collaborations.
For an in-depth overview of the possibilities of other types of sequencing, check out The Sequencing Buyer’s Guide report. It includes information about how to improve and reduce the cost of NGS workflows and explains the current patent landscape for sequencing.
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