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Base4 Reveals Details of Proposed Microdroplet Sequencing System

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NEW YORK (GenomeWeb) – Cambridge, UK-based company Base4 Innovation aims to develop a fast, affordable, and rapid sequencing scheme centered on microdroplet technology in parallel with the solid-state nanopore sequencing technology it is pursuing with Hitachi, Base4 Founder and CEO Cameron Frayling told In Sequence.

Earlier this year, Base4 announced that it had successfully identified individual DNA nucleotides — each carried in its own microdroplet — using a so-called cascade reaction that produces a distinct fluorescent signal from each of the four bases.

After capturing each unmodified nucleotide in a droplet, the cascade reaction "converts single nucleotides or droplets into a bright fluorescent signal," Frayling said. To sequence these bases, he continued, the team would need to keep these microdroplets in order and track their signals within a channel, while adding in more channels to bump up the throughput.

The company is pursuing the microdroplet sequencing strategy independently of the collaboration it entered into with Hitachi last summer. That effort centers on a solid-state nanopore system that uses gold nanostructures that sit near the nanopore to help produce signals that can be detected optically, rather than reading electrical signals as individual bases pass through the pore.

In contrast, the microdroplet approach involves an enzymatic process known as pyrophosphorolysis that uses pyrophosphate addition to prompt a polymerase enzyme to work backwards and release individual nucleotides from an immobilized, single-stranded DNA molecule.

Once liberated from the non-amplified DNA strand, these individual bases are each encased in different microdroplets and successively moved through a micron-sized channel. From there, the cascade reaction prompts each nucleotide to produce its own, characteristic fluorescent signal.

Frayling said the Base4 team has demonstrated each step in this process independently in its in-house lab. Now, the firm is attempting to put all of these pieces together to produce a unified sequencing system.

"We can take a strand of DNA and immobilize it in a channel, we can degrade nucleotides from a molecule of DNA, we can capture those in droplets, and we can do base calling from that," he explained, noting that the final hurdle will be putting all of these elements together to produce sequences from a microdroplet system.

Frayling said that there have been challenges in maintaining the order of the microdroplets in the absence of an appropriate sequencing chip, since droplets had to be incubated separately. The Base4 team obtained a prototype microdroplet sequencing chip a few weeks ago, he added, and is now starting to take a crack at putting all of the steps together to generate sequences.

"We're testing [the chip] now and we expect to have our first sequences [in] two to six months," Frayling said. "I fully expect that there will be bugs to work out, but all of the elements that we've demonstrated independently have been put together and we're now getting all of them into a single chip."

Along with the individual base calling milestone it announced in May, Base4 has also made headway on other aspects of the microdroplet sequencing technology. For example, Frayling said the team dramatically enhanced the fluorescence signals associated with the DNA bases by tweaking the cascade reaction chemistry.

Consequently, he said, it now appears possible to achieve excitation using low-cost sources such as LEDs rather than multiple lasers. Moreover, the team believes it will be able to detect nucleotide-specific signals with consumer-level electronics resembling cameras used in existing tablet phone technology, instead of relying on more sensitive and costly camera systems.

Together, such advances are expected to dramatically curb the price of producing microdroplet-based systems in the future, Frayling said. "With the base calling and the breakthrough in the instrumentation cost, [the microdroplet method] is suddenly looking very, very attractive as a sequencing system."

The Cambridge team hopes to team up with some design and/or engineering companies in the future to accelerate the development of a commercially available sequencing instrument, Frayling noted, but is not currently collaborating with any other firms on the microdroplet sequencing project.

He said that the goal is to produce an instrument with a manufacturing cost that's as low as possible. If the firm is able to use consumer-level electronics as is now anticipated, for instance, the cost of producing a microdroplet-based instrument could be below $10,000.

Frayling did not speculate about the potential reagent costs, but said that his team hopes to achieve "the lowest reagent costs on the market" by performing the necessary reactions within microdroplets. Further development is also needed to establish an automated sample preparation protocol that fits with the type of applications researchers are interested in, he added.

At the moment, the continuous chemistry behind the microdroplet method takes about 40 minutes, excluding sample prep steps. Ideally, the team would like to cut the time needed for that process in half so that a sequencing run takes about around 20 minutes.

From Frayling's perspective, the Base4 microdroplet- and the solid-state nanopore sequencing strategies have their own potential advantages.

In the case of the microdroplet approach, the company is enthusiastic about the prospect of producing a fast, affordable instrument that can pump out long reads from single-stranded DNA with high base accuracy, even across tricky-to-sequence regions where the same nucleotide appears together many times.

These homopolymer sequences should not be a problem in the microdroplet system, according to Frayling, because each of the bases is trapped in different microdroplets that are separated by empty droplets and read independently.

Likewise, preliminary experiments suggest that the method is consistent regardless of the GC composition of a sample due to the space afforded between bases by the individual microdroplets.

"What people want now are high throughput, high accuracy, and low instrument costs," Frayling said. "I think with the microdroplet technology that's something that we can achieve."

On the other hand, he noted that the solid-state nanopore system that Base4 and Hitachi are developing could have an edge at some point in the future, since it shows promise for detecting not only DNA bases and cytosine methylation, but also a wide range of rarer DNA modifications that are missed by conventional sequencing methods.

"What I'd like to do is push the development of the microdroplet technology towards commercialization as fast as I possibly can," Frayling said.

"But I'm not going to give up nanopore development," he emphasized. "That will continue, but we need to find the right way to give it the time to develop."

Base4 remains privately held, but is looking at potential strategies for garnering funding in the future, Frayling said, including the possibility of going public at some point in the coming years. "We'd like to really accelerate development so we're looking at how we can do that," he said.

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