Decades of discovery set to revolutionise healthcare
1 Nov 2018
It can be argued that the development of any successful innovation that transforms healthcare would not be possible without the knowledge derived from decades of discovery science preceding it. Curiosity-driven discovery science provides the fertile ground upon which novel technologies for improving human health are developed; historical examples include the development of MRI for imaging, or radiotherapy for treating cancer. Similarly, nanopore sequencing - a novel technology with applications in healthcare - results from decades of different branches of interdisciplinary discovery science converging to produce a commercial sequencing device called the MinION that literally brings the power of modern genomics to the palm of your hand.
The development of nanopore sequencing is a triumphant example of how discovery science funded by the MRC was translated into a novel technology poised to deliver widespread societal impact. This technology enables hospitals, GP surgeries, or rural clinics to offer genetic sequencing ‘by the bedside’. Nanopore sequencing represents a paradigm shift in genomics and biotechnology.
The applications of nanopore sequencing are vast and ever-increasing.
Over the recent years, advances in technology have allowed many more diagnostic tests to be carried out at the patient’s bedside rather than the laboratory bench. Known as point-of-care testing, these technologies confer obvious advantages to both the patient and healthcare professionals; quicker results mean faster treatment, and less cost to the health service.
Until recently, routine point-of-care testing has been limited to biochemical tests such as the measurement of glucose or blood cell counts. But within the last decade, point of care diagnostic development has been expanding into the fields of infectious diseases and personalised medicine. The MinION sequencer, developed by Oxford Nanopore Technologies, is a tiny pocket-sized DNA sequencer that performs nanopore-based sequencing in just a few hours. Launched in 2005, Oxford Nanopore Technologies was valued at £1.5 billion in May 2018 thanks to investment and expansion into the Asia Pacific region.
Nanopore sequencing is a ‘next generation’ DNA sequencing technique that uses a doughnut shaped protein whose hole is just a millionth of a meter wide – a nanopore.
These key milestones include;
One of the key techniques that was essential for revealing the structure of DNA was known as ‘X ray crystallography’. By passing a beam of X rays through a crystal, scientists can analyse the resulting diffraction pattern to work out what the structure of that crystal is. It’s like shining a torch at a hidden chandelier, and then working out its shape from the pattern of spots it makes on the wall.
Realising the potential applications of X ray crystallography, in 1946 we set up a new Biophysics Research Unit at Kings College London. A year later, in 1947 we also set up a Unit for Research on the Molecular Structure of Biological Systems in Cambridge (later renamed as the MRC Laboratory of Molecular Biology) to enable the study of proteins using X-ray diffraction to study proteins.
This early strategic investment into the newly developing field of structural biology paid off within a few years; scientists Dr James Watson and Professor Francis Crick were recruited to the MRC Molecular Structure of Biological Systems Unit while Professor Maurice Wilkins and Dr Rosalind Franklin began work at the MRC Biophysics Research Unit. Together, the work of these four scientists showed how four chemical letters A, T, C and G interact in three dimensions to form a long spiralling molecule with a double ‘backbone’ made up of sugar and phosphate blocks. Nitrogen-containing compounds, called bases, protrude from the two halves of the backbone and link together in pairs, so the whole molecule is like a zip.
The insights and new technologies spawned from the now famous double helix structure of DNA fundamentally changed the landscape of cellular biology, and heralded the golden age of molecular biology. The double helix has even become pervasive in art and design, a testament to its assimilation into the public consciousness.
Each living cell is surrounded by a membrane, and embedded into this membrane are protein channels through which the cell interacts with its external environment. Some of these channels can open and close to selectively allow the passage of molecules which are made up of charged atoms, or ions. Some membrane proteins - like α-hemolysin used in nanopore sequencing - are open all the time, allowing a continuous flow of ions through the pore. The flow of ions through a membrane pore can be read as a current flow, much like how an electric current through a wire is a flow of electric charge. These discoveries and techniques revolutionised molecular biology and electrophysiology, and decades later, aided the development of a single-molecule detection technology known as stochastic sensing, which was instrumental for the development of nanopore sequencing.
The first quantitative description of a current through a cell membrane channel was reported in 1970 by Dr Hladky and Professor Haydon. They reconstituted a bacterial ion channel into an artificial membrane and measured the current produced when the channel spontaneously opened. Six years later in 1976, Professor Neher and Dr Sakmann developed a technique known as the patch-clamp and successfully recorded the current flowing through a single channel in a biological membrane, and were awarded the 1991 Nobel Prize for Physiology.
Although the structure of DNA was revealed by Dr James Watson and Professor Francis Crick in the early 1950s, sequencing DNA molecules (i.e. determining the order of the four chemical letters A, T, C and G within the long spiralling molecule of DNA) was not yet a reality by the early 1970s. This was because DNA molecules are extremely long, and researchers had not yet found an enzyme capable of cleaving the DNA into smaller, more manageable pieces.
In 1962 Dr Sanger joined the MRC Laboratory of Molecular Biology as the Head of the Protein and Nucleic Acid Chemistry Division and soon turned his attention to the problem of sequencing nucleic acids such as DNA. In 1977, Dr Sanger and his team introduced an improved method for sequencing DNA, known as the ‘dideoxy chain termination method’ or the ‘Sanger method’. The importance of this work was recognised a short time later in 1980, when he was awarded the Nobel Prize in Chemistry. The Sanger method would go on to shape the fledgling field of genomics, and was instrumental to the Human Genome Project, the international collaboration to identify all the approximately 20,000-25,000 genes in human DNA.
The ‘central dogma of molecular biology’, as postulated by Professor Francis Crick in 1957 outlined how genetic information flows from DNA to proteins. This genetic code - programmed into the DNA molecule - determines the number and sequence of amino acids in a protein, and therefore the functional properties of that protein. By reprogramming the code of a DNA molecule, for example changing the word CAC to GAC, it would be possible to obtain a protein in which the amino acid histidine is replaced by the amino acid aspartic acid. In doing so, the shape and structure of the entire protein molecule can potentially be affected, because histidine carries a positive charge while aspartic acid is negatively charged. This can in turn result in the construction of a protein with desirable properties such as solubility or membrane binding.
In 1975, Dr Smith undertook a sabbatical year at the MRC Laboratory of Molecular Biology (MRC LMB) in Dr Fred Sanger’s lab. Sabbaticals allow scientists the opportunity to work in intellectually stimulating new environments while developing new techniques and collaborations. The MRC LMB was considered one of the leading institutions in the rapidly developing field of molecular biology, and the free-flow of ideas and people was characteristic of the time. The sabbatical at the MRC LMB provided Dr Smith with the background and inspiration that led him to the recognise that changing a single base in a gene could lead to a protein substituted amino acid. In 1978, in collaboration with another former Sanger lab sabbatical colleague Professor Hutchison, he introduced a new technique known as "site-directed mutagenesis" into molecular biology.
Over 20 years later, Professor Hagan Bayley used site directed mutagenesis techniques to modify membrane proteins for the application of nanopore sequencing. Site-directed mutagenesis presented an invaluable opportunity to manipulate the fundamental building blocks of DNA and proteins, and in recognition of this contribution, Dr Smith was awarded the 1993 Nobel Prize for chemistry.
Nanopore sequencing was pioneered by Professor Bayley, starting in the early 1980s when his team began studying the structure of a doughnut shaped protein called α-hemolysin in Staphylococcus aureus bacteria. S. aureus is a common bacterium responsible for causing a wide range of infections by destroying red blood cells via the α-hemolysin protein; α-hemolysin forms a pore on the red blood cell’s membrane and perforates it, causing it to leak its contents. At the time when Professor Bayley and his team were studying the structure of α-hemolysin, the possibility of nanopore sequencing was not on their agenda. However, by the late 1980s thanks to nearly 40 years of DNA research and the development of techniques such as single ion channel recording, it was clear that an artificial membrane with embedded α-hemolysin nanopores could be used for sequencing DNA.
As Professor Bayley observed these membranes containing pore proteins, he noticed that it was possible to measure and identify the molecules that pass through the nanopore. All cells have a membrane that separates the interior of the cell from the external environment. Given that many biological molecules carry small electrical charges, there is a difference in electrical potential between the inside and the outside of the cell. This membrane potential can be used by pore proteins that span the cell membrane to allow the flow of charged molecules from one side to the other. In the case of the α-hemolysin pore, it allows a steady measurable ionic current through its inside channel. Because DNA is a charged molecule, it is drawn through this open channel due to the difference in membrane potential. The entry of the DNA molecule into the pore gives rise to temporary, measurable variations in the current that are characteristic of the molecule passing through; these can be measured and used to identify the molecules involved.
In 2003 Professor Bayley moved to the UK and joined the Department of Chemistry at the University of Oxford. Supported by funding from the MRC, BBSRC, and EPSRC, his work on nanopore technology continued to thrive alongside a rapidly expanding research team. Building on techniques and discoveries made by fellow scientists, his research led to many breakthrough developments, particularly in techniques that allowed the ‘sensing’ of individual molecules passing through a pore. Nanopore DNA sequencing was born, and in 2005, Oxford Nanopore Technologies Ltd (ONT) was formed to support and further translate his research into applications in sequencing. Since its formation, ONT has raised over £250 million in investments and is valued at £1.5 billion (May 2018). ONT has also developed collaborations with leading nanopore researchers at other institutions, giving the company its market-leading position in nanopore sequencing.
In 1996 scientists showed that a single DNA molecule could move through a protein nanopore embedded within a biological membrane. However, the DNA molecule moved through the protein nanopore extremely fast; in the range of 1-10 microseconds per DNA base. This speed was too fast to accurately and reliably identify the sequence of the bases forming the DNA molecule as it moved through the pore; it was like trying to identify the passengers on a high-speed train by standing near the entrance of a tunnel as the train rushed past. In 2008, scientists at the Scripps Research Institute in the US, in collaboration with Professor Hagan Bayley, solved this problem by using an enzyme called DNA polymerase to slow down the DNA molecule as it passed through the protein nanopore. The DNA molecule moved through the protein pore at a much slower speed of around 2 milliseconds, a single base at a time. The single-base resolution of this approach and the ability to control the passage of DNA in single-base steps allowed nanopore sequencing technology to become a reality.
- In 1998, US and UK researchers including Dr Sydney Brenner and Sir John Sulston finished sequencing the genome of a tiny transparent nematode worm called Caenorhabditis elegans. The C. elegans genome project was the first complete genome of a multicellular organism.
- In 2001 an international team of scientists including researchers funded by the MRC and the Wellcome Trust published an initial draft sequence of the human genome.
The cost of sequencing genomes has fallen dramatically, while the speed and accuracy of the next generation of sequencing technologies has increased.
Of all the products developed by ONT, the MinION DNA sequencer is perhaps the most well-known. It is tiny, portable, and relatively cheap thanks to being the size and weight of a chocolate bar, powered by a laptop USB cable, and costing $1,000. For comparison, a DNA sequencing machine produced by a rival company using different technology, is the size of a washing machine and costs about $750,000. The MinION DNA sequencer quite literally brings the power of modern genomics to the palm of your hand; it’s small size and low cost means that it can be used in a variety of settings, ranging from small clinics to the field.
A recent study published in 2016 assessed the MinION’s sequencing performance against two machines currently available on the market and reported good agreement on accuracy across all three technologies. The MinION platform is under continuous improvement thanks to continuous field-testing by researchers and the spirit of open development embraced by ONT; sequencing data, software improvements and sample preparation protocols are being shared on the internet between researchers, forming a ‘nanopore sequencing community’.
The West African Ebola virus outbreak in 2013 was the largest on record, responsible for nearly 30,000 infections and almost 12,000 deaths. Because the Ebola virus has a rapid but constant mutation rate, during a prolonged epidemic it is possible to identify distinct sub-lineages of the virus. By sequencing Ebola genomes from newly diagnosed patients, the team could chart the source of new infections. If two people had identical strains of the virus, it was likely that they infected each other or at least they are nearby links on the same transmission chain. By attributing fresh diagnoses to existing clusters of the disease, the team could help local health workers identify the routes through which the virus was spreading, and develop effective strategies for stopping it.
However, staying one step ahead of the Ebola virus meant that speed was of the essence. Until then, sequencing Ebola genomes required a laborious process of sending clinical samples to specialist labs with expensive sequencing equipment that took weeks or even months to produce results. In contrast, the MinION sequencers used by the team helped them produce results less than 24 hours after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 minutes.
Real-time genomic surveillance is an invaluable new tool in our arsenal as we battle global disease outbreaks such as Ebola virus. Swift and accurate genomic information helps improve resource allocation and the timeliness of epidemiological investigations. Rapid sequencing of viral genomes also helps identify previously unknown routes of transmission, for example through food vendors or frequent travellers across borders. The design of a genomic surveillance system using MinION sequencers for infectious diseases can be easily adapted for other pathogens, and indeed the 2013 Ebola virus outbreak helped develop a blueprint for deploying the MinION sequencers for the 2016 Zika virus outbreak.
In early 2015, a widespread epidemic of Zika fever in Brazil spread to other parts of South and North America. In January 2016, the World Health Organization (WHO) said the virus was likely to spread throughout most of the Americas by the end of the year. Because Zika virus infection during pregnancy was linked to birth defects including brain damage in thousands of babies, addressing the Zika epidemic became a global priority.
Viral genomic sequencing is an important tool for tackling emerging epidemics, and sampling the viral genome allows researchers to quantify the genetic diversity of the virus, reconstruct its origins, estimate rates of transmission while providing background information for vaccine development and drug design. However, until July 2016 only 23 Zika virus genome sequences from Brazil were publicly available, despite over 190,000 suspected Zika cases being reported to the Brazilian Ministry of Health since 2015. This lack of Zika virus genome sequences resulted in an extremely fragmented picture of the diversity of Zika virus circulating within Brazil, and made it difficult for researchers to develop a vaccine or design new drugs for preventing or treating the infection.
A UK-Brazil collaboration, part funded by the MRC Zika Rapid Response Initiative successfully piloted the portable MinION DNA sequencer in the field to directly sequence Zika virus from clinical samples. Previous work in 2013 by Professor Nick Loman and his team in monitoring the spread of the Ebola virus outbreak in West Africa was particularly useful for designing a genomic surveillance system for Zika virus in South America. During the project the researchers trained Brazilian fieldworkers to perform sequencing using the MinION platform, and sequenced over a thousand clinical samples. As part of the Zika Rapid Response Initiative’s commitment to open science practices, both the protocol and materials used are freely available through the ZiBRA project website. These results provided a framework for reconstructing the trajectory of the Zika virus spread through the Americas and helped track its spread into other geographic regions. The MinION sequencer is ideally suited for developing a surveillance network capable of detecting and characterising viral spread in real-time during a public health emergency.
In 2016, 10.4 million people fell ill with TB, and 1.7 million died from the disease (including 400,000 among people with HIV) in the world. Over 95% of TB deaths occur in low- and middle-income countries. Although antibiotics to treat TB have been available since the 1950s, the drugs must be taken in complicated combinations for many months to be effective. First-line TB treatment includes four drugs (rifampin, isoniazid, ethambutol, and pyrazinamide), but with the spread of multidrug-resistant strains, there is a growing need for data on second-line drugs, including the fluoroquinolones and aminoglycosides. Thus, forecasting whether a strain of TB could be killed by a certain drug, i.e. predicting its drug susceptibility by analysing whether a patient has a drug-resistant strain of TB is very important.
In 2017 a team led by Dr Zamin Iqbal at the University of Oxford published the results of their work developing a same-day diagnostic test by sequencing sputum samples from patients with TB using the MinION sequencer. The test will also predict drug-susceptibility and inform disease surveillance by demonstrating the genomic relationships to previously seen strains. The team won seed funding from the Longitude Prize Discovery Award, and are hoping to do a pilot study of their diagnostic in Ho Chi Minh City and Mumbai, two cities with a high level of multi-drug-resistant TB - therefore a large TB burden. The handheld MinION sequencer has already proven that it can get extremely accurate results, and if rapid comprehensive tests become widely available in these vulnerable regions of the world, it could drastically reduce the amount of undiagnosed drug resistant TB.
Currently the gold standard diagnostic method for TB diagnosis is through laboratory culture. However, as the M. tuberculosis bacteria grow and divide very slowly, this process can take up to 12 weeks to deliver results. In addition, a considerable proportion of the TB cases reported to WHO are still clinically diagnosed rather than bacteriologically confirmed. In 2016, for example, only 57% of the pulmonary cases reported to WHO were bacteriologically confirmed. All this highlights the urgent need for a rapid, portable, easy-to-use diagnostic method that can be used at the patient’s bedside. The diagnostic method should be capable of identifying the presence, strain type, and any drug resistance of M. tuberculosis bacteria in patient samples, while also providing information on drug susceptibility and enabling disease surveillance. This is the first step in revolutionising the management of TB so that global eradication can become a reality.
In May 2018, researchers at the University of Nottingham and Queen Mary University of London achieved a new milestone in genomics by sequencing a single DNA strand more than two million bases (2 Mb) in length using the MinION sequencer. Other competing genome sequencing technologies have not been able to sequence DNA strands of this length and breaking the 1 Mb barrier is therefore considered a milestone. Sequencing a single DNA strand of more than 2 Mb is akin to running a 6.4-kilometre piece of rope through a fist, at a speed of about 1.5 meters per second. Another useful analogy to understand the vast quantity of data that resides within our genome is, if the genome were represented by the entire human population, then this 1 Mb long read would encompass the entire population of Birmingham and Manchester combined, while a single read from a traditional sequencing platform would correspond to the number of people in a busy train carriage. It’s easy to see why the speed and efficiency of nanopore sequencing distinguishes it from traditional sequencing methods.
Another advantage of nanopore sequencing technology is the ability to sequence epigenetic modifications. Epigenetic modifications are heritable markers that are not due to changes in DNA sequence. Rather, epigenetic modifications involve a chemical “tag” such as a methyl group being attached to bases in the DNA strand which alters the accessibility of the DNA, thereby regulating how and when genes are expressed. Traditional DNA sequencing technologies cannot directly distinguish between methylated and unmethylated bases in a DNA strand without specialised sample preparation. In 2017, scientists showed that the MinION sequencer can detect epigenetic modifications without this specialised preparation. This exciting development opens many research possibilities by allowing both the genome and the epigenome to be analysed from a single sequencing run.
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