March 1, 2018 (Vol. 38, No. 5)
Portable, Low-Cost, Real-Time Nanopore Instrument Democratizes Genome Analysis
When the Ebola virus spread through West Africa a few years ago, a new, portable DNA sequencer developed by Oxford Nanopore Technologies (ONT) was brought to New Guinea. There, the sequencer analyzed samples of the virus and provided near-real-time results giving researchers clues to how the disease spread.
In addition, the same model of sequencer was transported in a mobile laboratory (on a bus) around Brazil, where it was used to build a family tree for the Zika virus, identifying a large number of strains and, surprisingly, indicating that the virus had been in the country much longer than anyone had realized. “No other technology allows you to do that (in the field),” Gordon Sanghera, Ph.D., CEO, ONT, says.
As part of that project, researchers in Brazil formed a consortium, called the ZiBRA project, in collaboration with Instituto Evandro Chagas and Fundação Oswaldo Cruz public health laboratories in Brazil, to bring mobile, full-genome sequencing to monitor Zika. Results of the monitoring are discussed openly on the Virological.org forum and are expected to help manage public health crises.
The ability for any lab to sequence the genome of a pathogen quickly and cost-effectively is a game-changer for public health. “This is an important tool in the surveillance and control of infectious diseases,” Dr. Sanghera emphasizes. “It’s in the very early days, but real-time DNA sequencing is a significant step forward.”
It’s a step forward for drug developers, too. For biotech, process monitoring for biologics is an obvious application with quantifiable payback. For example, Boston-based biotech Ginkgo Bioworks’ use of the MinION for real-time sequencing alerted operators to changes within 8 hours versus the 18–30 hours typically required. That let the company determine the contaminant was nontoxic and filter it out, thereby saving the batch. In the process, the company saved approximately $100,000, according to an online presentation delivered by Rachel Rubenstein, who is currently an NGS intern at Ginkgo Bioworks.
Other companies are interested in sequencing to understand antimicrobial resistance, identify airborne pathogens and bioterror threats, and elucidate the relationship between a gene mutation and disease.
Other industries will benefit, too. Some of ONT’s early use cases have been in the food industry, where one chocolate company uses the MinION to look for signs of Salmonella within its supply chain. A portable sequencer could also assess food quality and help municipal water facilities check reservoirs for contamination. There are many applications for an inexpensive, deployable sequencer, according to Rubenstein. A larger version of the MinION instrument, the GridION, is even being used to sequence the genome of the tulip—at 34 Gb, it is one of the largest genomes around.
“Whether we’re talking about manufacturing biologics, processing food, or purifying water, once you have the ability to look at DNA in real time, you change the monitoring paradigm,” Dr. Sanghera points out. This is an opportunity, he continues, for organizations to change their missions or adjust production processes.
This sequencing approach is based upon passing ionic current through protein nanopores that are embedded into a polymer membrane. As the bases pass through the nanopore, the base sequence is decoded in real time. The results can be delivered to users immediately, rather than after hours or days, as with traditional sequencers.
“This is quite a mindset shift,” Dr. Sanghera admits. “Conventional DNA sequencers are fast, but not fast enough.” They offer quasi-real-time sequencing in 18–36 hours. “But, because they multiplex batches of samples, you need a large number of samples,” he adds. “With MinION, you can sequence samples at any point [in the] manufacturing [process] in real time without going to a lab.” Consequently, users can minimize lost opportunity costs.
Democratization of Sequencing
ONT’s MinION sequencer is about the size of an audio recorder. It slips into a pocket easily, offers 512 nanopore channels, prepares samples in 10 minutes, and costs only $1,000 for a starter pack. It sequences the length of a DNA fragment that the researcher prepares, resulting in reads of any size from a few hundred bases to the current record of 1.3 Mb. Initial results are returned within minutes.
That combination of speed, portability, and affordability puts real-time, whole-genome sequencing within reach of any scientist in the world. Because the device operates in real time and can be reused, it heralds a new paradigm. “There’s no need to wait for samples to accumulate for multiplexing, or to send samples to a distant lab for sequencing and analysis,” declares Dr. Sanghera. “Scientists can run samples cost-effectively when they need them, even if that means running a single sample.”
During the early 2000s, Dr. Sanghera was scouting for new, potentially disruptive technologies. Oxford University’s innovation center became a favorite haunt. That’s where he met his cofounders, Spike Willcocks, D.Phil., and Hagan Bayley, Ph.D. “I realized,” Dr. Sanghera recalls, “I could apply my expertise in bioelectronic glucose monitoring (gained at Oxford spinout MediSense) to Professor Bayley’s work and develop a disruptive, paradigm-shifting technology for DNA sequencing.”
Establishing proof of concept was his first priority. “I took a glucose sensor, cut it up, took out the bits I needed, and did a live proof of concept of nanopore sensing before we founded the company (in 2005),” he details.
The early challenge was raising money for what would become an eight-year-long endeavor. After four years, ONT’s scientists had achieved a significant breakthrough in the form of exonuclease sequencing technology. Two years later, they changed paradigms to pursue a strand-sequencing approach.
“That was a difficult period, when we shifted chemistry,” Dr. Sanghera admits. Years later, he came to liken the building a new company—particularly one with a disruptive technology—to raising a child: “We’re through the ‘broody twos’ now.”
MinION was introduced for select, early access partners in 2014. And now, Dr. Sanghera insists, “Manufacturing is stable, we’re scaling the business and our commercial execution, and we’re in the foothills of commercialization.” Today, the primary challenge when talking with investors is the comparison to large, mainframe systems. “We’re not a mainframe company,” he emphasizes. Therefore, the users and value proposition are different.
“For MinION, many of our users are people who would be lost to the mainframe sequencer market. They want to do sequencing but lack the substantial capital budget. Our approach, using MinION, is opening new markets and democratizing sequencing,” he says.
A Busy 2018
Although portable sequencing is important, high-throughput users aren’t ignored. The company’s newest sequencers, GridION and PromethION, are designed for those users. The GridIONx5, a desktop sequencer, launched in 2017. It processes up to five MinION flow cells at a time, offering up to 2,560 nanopore channels.
This year, ONT plans to launch the PromethION for large, population-scale sequencing projects or larger numbers of samples for on-demand sequencing. In internal testing, PromethION recently has been generating 90–120 Gb on a single flow cell. PromethION currently is designed to offer up to 48 flow cells, each with 3,000 nanopore channels (totaling 144,000 channels), though the throughput of the earliest versions may be lower.
Geographic expansion is progressing, too. The company’s products are available in more than 70 countries, and ONT-certified laboratories offering ONT’s technology are now operating in Europe, the United States, China, and Australia.
By using ONT's sequencing approach, startup biotechs and public health facilities have access to the same whole-genome sequencing data that otherwise would be the purview of labs that are larger or have deeper pockets. Today, collecting genomic information no longer needs to be a waiting game.
Oxford Nanopore Technologies