Photograph of Professor David Deamer with his second wife, Professor Ólöf Einarsdóttir, a biochemist, shortly after they married in 1992 (credit David Deamer). Brought up in California and then Ohio, Deamer completed a doctorate in lipid biochemistry at Ohio State University School of Medicine in 1965 and then spent two years at the University of California, Berkeley as a postdoctoral researcher in the laboratory of Lester Packer. Following his stint at Berkeley, Deamer joined the biological sciences faculty at the University of California, Davis where he remained until 1994 when he moved his lab to the University of California, Santa Cruz (UCSC).

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Definition

Nanopore sequencing makes it possible to sequence nucleic acids – DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) – directly from biological samples in real time. Approximately a quarter of all SARS-CoV-2 virus genomes sequenced worldwide to date have been done on a nanopore device. One of the latest generation in sequencing technologies, the technique determines the order of nucleotides in DNA or in RNA by measuring fluctuations in an electric current as the molecule passes through a nanopore. The nanopore, a tiny hole one billionth of a meter in diameter, is embedded in a membrane that separates two chambers containing electrolyte solutions. When a small voltage is applied, an enzyme steadily ratchets the molecule through the nanopore along with an ionic current. Specialised software works out its sequence based on how much short sequences of individual nucleotides block the flow of ions and tiny changes in electrical current. Both DNA and RNA contain adenosine (A), cytosine (C) and guanine (G) nucleotides. They also differ by one nucleotide. DNA has thymine (T), while RNA has uracil (U).

Importance

Nanopore sequencing is rapidly becoming one of the fastest and least expensive methods for deciphering genetic variations at the molecular level. First released on to the market in 2014, the method has radically transformed the way sequencing is carried out. Unlike previous forms of sequencing nanopore sequencing does not require a sample to be initially amplified to carry out the sequencing. Moreover, it can sequence a complete nucleic acid strand in one go, sometimes over a million bases in length, so sequences no longer need to be assembled computationally from fragments just a few hundred bases in length. It also has the benefit that it can read a sequence directly from a biological sample in real-time.

Now available in highly portable devices which can be plugged into laptops to upload data to the cloud, the technology makes it possible to carry out sequencing for the first time in remote areas with limited laboratory resources, no internet access or electricity supply. It also does not need any specially trained staff. This makes nanopore sequencing an invaluable tool for the rapid identification and monitoring of pathogens responsible for new disease outbreaks as well as tracking chains of transmission. Pocket-sized nanopore sequencers, for example, enabled viral genome sequences to be rapidly obtained from blood samples taken from patients during the outbreaks of the Ebola virus in remote areas of West Africa and the Zika virus in the hard to reach forested regions of Brazil. More recently, the technology was used in China to sequence and identify SARS-CoV-2, the virus causing the COVID-19 pandemic, and its variants.

Virus outbreaks are just the start of where researchers have begun to demonstrate the utility of nanopore sequencing. What is particularly attractive about nanopore sequencing is it offers results in real-time and it can sequence both DNA and RNA. The possibility of sequencing RNA was first flagged up by in 2012. One of the advantages of using the technology to sequence RNA is that it circumvents the need for reverse transcription or amplification steps which other methods require (Garalde et al).

Aside from viruses, nanopore sequencing provides a major tool for the detection and control of bacterial infections resistant to antibiotics, which is a major growing threat to global public health. Able to rapidly identify a microbe, nanopore sequencing could soon supplant the need for the time-consuming process of culturing bacteria for identification and testing their susceptibility to antibiotics, thereby enabling physicians to shift away from the use of broad spectrum antibiotics to more specific treatment plans. Nanopore sequencers could thus become essential to the good stewardship of antibiotics and reducing the over-prescription or futile use of such drugs.

In addition to improving the diagnosis and treatment of patients, nanopore sequencing provides a means to map out the evolution of many different pathogenic microbes. It can also help track their transmission routes, which is vital to the surveillance of emerging infectious diseases and bringing epidemics under control. Nanopore sequencing has many more applications, including in fundamental biological research, ancestry analysis, and forensic science. The technology is now being used for example to explore microbes found in the most extreme and inaccessible environments on Earth, such as in the Arctic and Antarctic (Edwards et al 2019; Johnson et al) and a deep mine in South Wales (Edwards et al 2017).

When nanopore sequencing was first released to researchers in 2014, it was used to read bacterial and viral genomes, or for interrogating small targeted regions within the human genome. Since then a number of advances have been made which has enabled the reading of much larger genomes, opening up its potential use for human genomes (Bowden). The first human genome sequenced with nanopore sequencing was reported in January 2018. This was a major turning point for the technology because the human genome is used as a yardstick to assess the ‘performance of DNA sequencing instruments’ and is now an important tool in medicine, such as in cancer research and diagnosis (Jain et al).

Discovery

Many of the scientists behind the development of nanopore sequencing point out that its emergence was gradual and rested on the coming together of several techniques developed by different players based in both academia and industry. Nanopore sequencing therefore cannot be seen as a sudden eureka breakthrough by one individual working alone or as a linear process. Nor was its development simple and quick. Instead it was a gradual process which involved multiple academic and industrial actors, each contributing to important aspects of its development from different directions and disciplines. Each of the participants behind its evolution have their own story to tell.

The history of nanopore sequencing is also an important reminder of the crucial role of collaboration between academia and industry. What contributions each made can be traced in different ways. While publications help reveal the path taken by academics, patents provide a map of the work undertaken by the commercial scientists (Bayley interview; Brown emails; Brown, Clarke, Willcocks interview).

One of the major foundations for nanopore sequencing was the development of single-channel recording, a technique developed by Erwin Neher and Bert Sakmann in 1976, respectively based at Yale University School of Medicine and the Max Planck Institute for Biophysical Chemistry in Göttingen. Awarded the Nobel Prize in 1996, Neher and Sakman developed a means to study ion permeation mechanisms in biological membranes. They did this by clamping a micropipette, or patch pipette, to the membrane filled with an electrolyte solution, connected to an electrode and placing another electrode in a bath surrounding the cell or tissue to form an electrical circuit. Refined over the years, Neher and Sakman’s method provided a means to record and measure the amount of current flowing through a single ion channel when it opens and closes (Neher, Sakmann; Bayley, Martin).

Nanopore sequencing begins as an idea

David Deamer, an American biologist and biomolecular engineer was one of the first people to propose using a nanopore to sequence DNA. In 1989, while based at the University of California, Davis (UC Davis), Deamer sketched out a plan to use an electric field to drive a single-stranded DNA molecule through a protein nanopore incorporated into a very thin membrane made up of two layers of lipid molecules, known as a bilayer (Bayley, 2015). Knowing that individual nucleotides, the small units of DNA, differed in size, Deamer hypothesised that it would be possible to work out their sequence by measuring the extent to which they blocked an ionic current as they passed through the nanopore (Deamer, Akeson, 2000. ).

Being able to synthesise RNA from scratch was an important component of Deamer’s wider research to understand how the first forms of primitive life came to exist on earth. Deamer had first become interested in how life began in 1975 during a sabbatical he spent with Alec Bangham at the Animal Physiology Institute in Babraham, a few miles south of Cambridge University. Bangham, a British biophysicist and haematologist who had a strong interest in the physicochemical properties of membranes and cell surfaces, was best known for his discovery of liposomes in 1961 (Watts). Inspired by his conversations with Bangham, Deamer decided to focus his research on investigating the role of membranes in the origins of cellular life (Damer).

As well as attempting to synthesise RNA in a liposome, Deamer had another line of research that helped him formulate his nanopore sequencing idea. This was directed towards understanding the molecular basis of transport in biological membranes, the process by which a substance gets transferred across a cell membrane (Deamer, 1992). He was doing this work with Mark Akeson, a postdoctoral researcher in his laboratory. Their project was focused on investigating different types of channels that could enable solutes like potassium ions and hydrogen ions to pass through lipid bilayer membranes. One of these was gramicidin, an antibiotic peptide produced by the bacteria species Bacillus brevis. Gramicidin forms very small ion channel-like pores in the cell membrane. From this work Deamer knew that it was possible to get substances to travel across membranes by using a protein to create a channel in the membrane (Deamer interviews).

Initially, Deamer viewed the ion channel (nanopore) created by gramicidin as a means to deliver ATP into a liposome to synthesise RNA. It did not take long for him to realise, however, that if he was able to thread ATP, a large molecule, through a nanopore in a membrane with the help of an electric field, it might also be possible to do the same with a string of nucleotides in a piece of DNA (Deamer interviews).

Deamer envisioned nanopore sequencing working along the same lines as the Coulter counter, an instrument routinely used to count blood cells and other particles (Deamer interviews). Invented in 1954-55, the Coulter counter consists of a tube with a very small hole in its wall which is immersed in a beaker with particles suspended in an electrolyte solution. The hole acts as a sensing zone. Electrodes are placed both inside and outside the tube to form an electrical circuit. The number of cells and particles are counted by measuring transient changes in the electrical current as they pass through the hole (Beckman Coulter).

The nanopore sequencing method proposed by Deamer was radically different from the DNA sequencing approach first described by Fred Sanger, in 1977, for which he went on to win a Nobel Prize. Widely commercialised and used by laboratories around the world, Sanger’s method works by using a polymerase, an enzyme, to synthesize DNA and the addition of tags to distinguish DNA fragments with different nucleobase endings. These fragments are then placed on acrylamide gels in different lanes and an electric current is applied which separates the fragments according to their size. The sequence is deduced from black bands read from an x-ray film which captures the position of the nucleotides on each of the fragments. By the late 1980s the Sanger technique had become an automated process, but it remained slow and expensive for determining the sequence of large and complex genomes like those of humans. This was because it required DNA in biological samples to be purified, amplified, fragmented into small pieces that were chemically labeled and then separated to read the dye labels. Before the sequence of the molecule could be worked out the fragments had to be reassembled by computational techniques. In principle, nanopore sequencing offered a much simpler and less expensive method (Branton, Deamer ; Deamer interviews).

Deamer could not pursue his idea immediately because he lacked a nanopore large enough to accommodate DNA. By 1991, however, he had learned about the research of John Kasianowicz, a physical scientist based at the National Institute of Standards and Technology in Gaithersburg, Maryland. Kasianowicz was experimenting with alpha-haemolysin, a protein toxin secreted by the bacteria Staphylococcus aureus (Branton, Deamer). The protein binds to the outer membrane of target cells in mammals, including humans, to form a water-filled pore. Uncontrolled permeation of vital molecules through the channel leads to osmotic swelling and the rupture of the cell membrane. In severe staphylococcal infections it is linked to the damage of red blood cells (NIH).

Kasianowicz was conducting investigations into alpha-haemolysin as part of a project he and colleagues had launched in the late 1980s to explore the physical properties of ion channels, proteins that form pores in membranes and enable ions – molecules with a net electrical charge – to flow across the cell membrane (Griffiths; Kasianowicz, Bezrukov). Alpha-haemolysin provided an ideal channel for Deamer to try out his idea. It formed a pore in lipid bilayer membranes just the right size for a single-stranded DNA molecule to pass through and was very stable and well behaved (Griffiths).

In 1991, Deamer took the opportunity to discuss the possibility of nanopore sequencing with Daniel Branton, a close colleague and friend from Harvard University when, at Deamer’s invitation, he gave a series of lectures at the UC Davis. During the visit the two scientists discussed ways to perform single molecule sequencing. Branton had been pondering the possibility of detecting individual bases in DNA by pulling a DNA strand through an interface (London Calling), but they decided that nanopore sequencing was a feasible alternative so began to collaborate on the project.

Being able to sequence DNA with a nanopore was highly attractive because the Human Genome Project had been launched the year before. Supported with public funds, this project aimed to sequence the entire human genome, made up of 3 billion nucleotide base pairs, within 15 years. This was a highly ambitious endeavour because until then the longest genome that had been sequenced was the Vaccinia virus, sixteen thousand times smaller than the human genome (Giani et al). In order to complete the human genome scientists needed a faster and less expensive means to carry out DNA sequencing (Branton, Deamer).

Once Branton returned to Harvard, he and Deamer began to look into applying for a joint patent between their two institutions. They decided to use the Harvard patent office to initiate the process because it had more resources to work on the application than the University of California. The Harvard office put them in contact with George Church, another Harvard faculty member who had disclosed a different idea to the office about nanopore sequencing. Church’s idea was to use a protein isolated from a bacteriophage to drive double- stranded DNA through its pore. Branton in fact knew Church. Having Church on board turned out to be highly fortuitous because the Harvard Patent Office staff were initially hesitant about pursuing a patent on nanopore sequencing. Part of their reluctance stemmed from their belief that nanopore sequencing was a ‘wild idea that was never going to work’. Church managed to change their minds after pointing out that if three professors from different laboratories had independently come up with the same concept then it must have merit (London Calling). Filed in March 1995, the first patent for nanopore sequencing was granted in August 1998. It incorporated both Deamer and Church’s techniques (US Patent 5,795,782).

With the patent application process initiated, in late 1992 Deamer flew to Gaithersburg to test out the haemolysin nanopore with Kasianowicz at NIST. Deamer took with him two forms of commercially available synthetic RNA which had been created with polynucleotide phosphorylase, the same enzyme he had previously used for his liposome experiment. One of the RNA’s was polyadenylic acid, composed of adenylic acid monomers, and the other was polyuridylic acid, composed of uridylic acid monomers. After setting up the experiment and inserting a single haemolysin channel into a planar lipid bilayer, Kasianowicz added one of the RNA species and began to increase the voltage across the membrane 10 millivolts at a time. They could see the ionic current increasing with each step, but nothing much happened until the voltage reached 100 and then 120 millivolts. Suddenly they began to see many transient blockades of the ionic current, each lasting for a few milliseconds. In some cases 90 per cent of the current was blocked. The same thing happened when they repeated the experiment with the other RNA species (Deamer emails).

The current blockades were just what they expected if long strands of the single stranded nucleic acid were being drawn through the pore by the applied voltage. If this experiment had not worked, the idea of nanopore sequencing would probably have been abandoned (Deamer interviews; Deamer emails). Excited by what he had witnessed, Deamer invited Branton to witness the next experiment. Conducted in March 1993, this was again successful. So encouraging was the result that Deamer and Branton decided to seek funding from the National Science Foundation’s small grants programme. The advantage of such a grant was that did not require peer review. For them the grant would help them resolve whether the blockades signified individual bases coming into contact with the pore as the DNA flowed through and was not resulting from a folded up wad of DNA colliding with the pore (London Calling).

Awarded $50,000 by the NSF, Deamer and Branton had enough money to work for two years with Kasianowicz (Deamer interviews). In 1994 the team repeated and extended their earlier experiments. They found that longer RNA polymers caused longer blockades of the ionic current as they passed through the nanopore than shorter polymers. The result was encouraging because it provided a means to count and characterise each nucleic acid (Deamer, Akeson, Branton, 2016a).

By 1996 the collaborators had sufficient evidence to demonstrate that an electrical field was able to drive single-stranded DNA and RNA molecules through the alpha-haemolysin pore but the double-stranded DNA double helix was too large. They also showed that the nanopore could unravel a coiled up nucleic acid so that its nucleotides flowed through the pore in a single-file sequential order. Another crucial observation they made was that the chemical nature of the nucleotides – purines (A, G) or pyrimidines (C, T, U) – could be determined by how much they impeded the iconic current running through the pore (Branton, Deamer, Marziali).

Despite the team’s achievement, both Nature and Science, two highly prestigious scientific journals failed to recognise its relevance and declined to publish their findings. Eventually their article appeared in the Proceedings of the National Academy of Sciences (PNAS) in December 1996 (Kasianowicz et al 1996). Branton persuaded the journal to accept the article based on his membership of the National Academy of Sciences. According to Deamer a few months after the paper appeared, Science published a news report about a ‘remarkable new discovery’ that they had previously turned down as a manuscript (Deamer interviews).

The early experiments helped to establish that the nanopore could act as a sensor by measuring the ionic current blockades produced by RNA or DNA molecules. In 1997, Deamer obtained his first NIH funding for working on the project and persuaded Akeson to give up a full-time position to help him move it forward. The same year Branton also decided to spend a sabbatical with Deamer to join in on the work. The three scientists took the opportunity of being together to determine whether the alpha-haemolysin pore could distinguish between purine and pyrimidine bases (Deamer interviews).

Akeson met Deamer while he was doing a doctorate in soil microbiology at UC Davis. Having enjoyed a graduate course taught by Deamer, Akeson decided to join his laboratory as a post-doctoral researcher where he worked with Deamer on lipid bilayers until 1991 when he took up a position at NIH. In 1996 Akeson decided to rejoin Deamer, now at UC Santa Cruz, to collaborate on the research funded by NIH. For Akeson the move was a risky proposition because it required him to forgo a salaried position and work on what is called soft money provided by the grant. He also had no guarantee that his position would continue once the NIH grant ended. But Akeson had read the 1996 PNAS paper published by Deamer and his colleagues and decided the science was too exciting to turn down (Deamer emails).

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