Unravelling DNA

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Nadya Anscombe discusses the latest DNA sequencing methods, comparing optical with non-optical technologies

Just a few weeks ago, in mid-February 2016, the journal Nature announced that it will make all papers relating to the Zika virus free to access until further notice. It also encouraged authors who have not already deposited their relevant sequence information in public archives to do so on submission, to help drive the shift towards fast data sharing during public-health emergencies.

In the same week, in the same journal, a large global group of researchers published a paper entitled ‘Real-time, portable genome sequencing for Ebola surveillance’.1

Genome sequencing in viral outbreaks needs to take place locally and happen quickly, in order for it to be useful in characterising the infectious agent and determining its evolutionary rate. While DNA analysis can be used to determine whether a patient has a certain virus, DNA sequencing goes one step further and allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterisation of responses to vaccines and treatments.

One of the reasons that the recent Ebola outbreak in West Africa became the largest on record was that the virus mutated quickly. Genome sequencing can track the evolution of these mutations and be used to guide control measures, but only if the results are generated quickly enough to inform interventions.

But performing DNA sequencing in Africa is challenging for many reasons – there is a lack of local sequencing capacity, transporting samples to remote facilities is a challenge and the ambient climate also creates problems.

So a portable device that can reliably and quickly sequence DNA in the field has been a dream for researchers for many years.

In their Nature paper, the 104 authors from 13 different countries describe how they transported the MinION by Oxford Nanopore in standard airline luggage to Guinea, and how they were able to generate results less than 24 hours after receiving an Ebola-positive sample, with the sequencing process taking as little as 15 to 60 minutes. They showed that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

The MinION weighs less than 100g, is no bigger than an office stapler and plugs into a laptop using a USB cable. 

It works by taking frequent electrical current measurements as a single strand of DNA passes through a protein nanopore at 30 bases per second. DNA strands in the pore disrupts ionic flow, resulting in detectable changes in current that is dependent on the nucleotide sequence.

Unlike the majority of the leading lab-based DNA sequencing technology, the MinION relies on electrical, rather than optical technology. As this article went to press, there were no optical-based mobile DNA sequencing devices on the market, and, according to Dr Matthias Schulze, marketing director for Coherent, this situation is not likely to change in the near future.

‘The big players are concentrating on increasing the throughput of their products, and not on creating optical portable sequencing devices,’ said Schulze. ‘Optical technology is ideal for high-throughput and high accuracy and we believe laser-based systems will remain dominant as the industry moves to third-generation sequencing.’

But Schulze admitted that the industry needs to be prepared as optical technologies face an increasing amount of competition from non-optical technologies. ‘Costs have already come down substantially so, at the moment, high-throughput and not cost is the issue,’ he said. ‘But the optical community needs to be ready to face competition from non-optical technologies, when the technology concepts for sequencing continue to diversify to optimise tools in cost, packaging and performance for each application subsegment.’

The authors of the Nature paper admit that while the MinION is definitely portable, one piece of their kit was bulky – the thermocyclers that carried out the reverse transcriptase polymerase chain reaction (RT–PCR), an essential step in order to isolate sufficient DNA for sequencing.

Researchers in the US have come up with an optical solution not only to bring down the size of PCR equipment, but also to speed up the process.

‘PCR is powerful, and it is widely used in many fields, but existing PCR systems are relatively slow,’ said study senior author Luke Lee, a professor of bioengineering at UC Berkeley in the US. ‘It is usually done in a lab because the conventional heater used for this test requires a lot of power and is expensive. Because it can take an hour or longer to complete each test, it is not practical for use for point-of-care diagnostics.’

Conventional PCR is slow because of the time it takes to heat and cool the DNA solution. The PCR test requires repeated temperature changes – an average of 30 thermal cycles at three different temperatures – to amplify the genetic sequence, a process that involves breaking up the double-stranded DNA and binding the single strand with a matching primer. With each heating-cooling cycle, the amount of the DNA sample is doubled.

In order to speed up the process, Professor Lee and his colleagues took advantage of plasmonics, or the interaction between light and free electrons on a metal’s surface. When exposed to light, the free electrons get excited and begin to oscillate, generating heat. Once the light is off, the oscillations and the heating stop.

They used LEDs to heat electrons at the interface of thin films of gold and a DNA solution. They clocked the speed of heating the solution at around 55°F per second. The rate of cooling was equally impressive, coming in at about 43.9° per second.

For their experiments, the researchers used thin films of gold that were 120nm thick, or about the width of a rabies virus. The gold was deposited onto a plastic chip with microfluidic wells to hold the PCR mixture with the DNA sample.

The light source was an array of off-the-shelf LEDs positioned beneath the PCR wells. The peak wavelength of the blue LED light was 450nm, tuned to get the most efficient light-to-heat conversion.

The researchers were able to cycle from 131°F to 203°F 30 times in less than five minutes. They tested the ability of the photonic PCR system to amplify a sample of DNA, and found that the results compared well with conventional PCR tests.

‘This photonic PCR system is fast, sensitive and low-cost,’ said Professor Lee. ‘It can be integrated into an ultrafast genomic diagnostic chip, which we are developing for practical use in the field. Because this technology yields point-of-care results, we can use this in a wide range of settings, from rural Africa to a hospital ER.’

Professor Lee is now looking to mass manufacture his PCR technology and is currently in discussions with several companies. The genomic diagnostic chip that his group is developing also relies on optical technology. ‘Optical technologies for DNA analysis and sequencing have a well-established infrastructure, and electrical measurement is not as accurate as optical,’ said Professor Lee. ‘I think sequencing with optical technologies may be possible in the future, but it will require a huge team effort involving engineers, biologists and computer scientist working together.’

Professor Lee’s genomic diagnostic chip is one of many similar lab-on-a-chip technologies being developed around the world. Most research groups and companies will choose to go down either the optical or the non-optical path. But one company, UK clinical diagnostics firm QuantuMDx, is developing two technologies, one optical and one based on nanowire field-effect transistors (FET).

‘Which technology our customers use will depend on the kind of analysis they need to perform,’ said Jonathan O’Halloran, chief scientific officer of QuantuMDx. ‘Our FET technology has a higher sensitivity, but our optical technology is currently more affordable and more established.’

The company has managed to miniaturise the PCR component required for DNA analysis and integrate it into its Q-POC system. The system, which uses optimised peltier technology, can run at less than 10W and has amplification rates comparable with those of benchtop systems.

Its fluorescence-based technology is shortly entering a clinical trial to test patients for warfarin susceptibility. Warfarin, an anticoagulant prescribed for patients susceptible to blood clots, is metabolised by patients at different rates, so patients should be given different doses. Current practice is for doctors to prescribe a standard amount and then adjust the dose at a later date. ‘Our device can predict the correct dose by analysing the patient’s genotype,’ said O’Halloran. ‘Today’s blood tests take weeks to come back. Ours can be performed at the GP’s surgery in minutes.’ 

Both the company’s fluorescence technology and the FET-based technology can analyse samples without a preparation step. The fluorescence-based technology uses fluorescent markers and a miniaturised fluorescence microscope to analyse the sample. The FET technology uses an array of nanowires on a silicon chip. Probes are deposited on these nanowires and, when the DNA of interest binds with these probes, a change in resistance is detected.

The company’s Q-POC system can use both the fluorescence and the FET-based technologies for DNA analysis and diagnosis tests, but when it comes to sequencing DNA, O’Halloran admits that his company is developing the FET technology for this application.

‘We feel that, for sequencing using an optical system, the amount of optics required would be too cumbersome for a portable device,’ said O’Halloran. ‘We have shown that sequencing is possible on our FET technology using the sequencing-by-synthesis technique, but that is still a few years away from commercialisation.’

Another reason why the FET technology is the technology of choice for portable sequencing is its potential for cost reduction. As it is based on standard silicon processes, mass production will significantly reduce costs. This makes it ideal for QuantuMDx’ plans to target the developing world, where cost is a major issue.

‘In the early stages of our development, we received funding from the South African government,’ said O’Halloran. ‘It’s hard not to be motivated to develop a low-cost product when you spend time in poor townships and see how urgent is their need for quick diagnosis of infectious diseases.’

The Ebola epidemic was officially declared to be over on 14 January 2016. Just hours later, a new case of Ebola was confirmed in Sierra Leone and further flare ups are expected. With portable diagnostic and DNA sequencing technology being developed, the source of these flare ups can be found quickly by linking to previously infected individuals.

With companies and research groups around the world developing portable technology for DNA analysis and sequencing in the field, it is hoped that the next epidemic, whether it be Zika or another virus, can be contained and managed quickly.