This article has been updated from a previous version to include updated information about the extent of the Regeneron Genetics Center's sequencing program.
NEW YORK (GenomeWeb) – Formulatrix still hopes to expand the market for digital PCR — it just might happen a little later than the company originally expected.
Three years ago, the Boston-area company was gearing up to launch an inexpensive, plate-based digital PCR system called Constellation that it had hoped could bridge the gap between pricier, more complicated droplet-based digital PCR technologies and tried-and-true quantitative PCR platforms.
And while the company claims that early reception for the system was positive, it also received enough feedback on how it could be improved that it decided to completely reengineer the platform. Now, the company believes its next-generation system addresses many of the shortcomings of digital PCR technology in general, and will entice researchers to finally make the switch from qPCR.
"We developed [the original] product for a specific customer as kind of a one-off arrangement," said Thomas Rawlins, a marketing manager at Formulatrix. However, feedback from the mainstream market suggested that while the system was a great approach to digital PCR, the number of partitions it had did not provide enough dynamic range "So we … went back to the drawing board and, along with some additional market feedback about the throughput and workflow associated with current digital PCR offerings … designed a brand new product," he said.
Now, the company is getting ready to fully commercialize the next-gen product, still called Constellation, said Paul Ventura, the company's director of sales. In the meantime, Formulatrix has placed beta units in the hands of a few testers, including pharmaceutical firm Regeneron, and is still gathering feedback to make final tweaks to things like software and the system's user-friendliness. The company hopes to land one more cadre of early-access users by the end of this year, and fully launch the platform in the first quarter of 2018, Ventura said.
Perhaps the most important improvement to the system is that it is fully integrated and automated in one benchtop instrument, "so there is sample-to-answer, from partitioning to thermal cycling and imaging," Ventura noted. The workflow involves pipetting reaction mixture into a dPCR plate, manually applying a rubber plate seal, and inserting the plate into the benchtop instrument, which automatically partitions the samples, performs thermal cycling, and reads the plate.
The company plans to sell two models: one that can run four plates and one that can run eight plates simultaneously. It takes about 90 minutes to produce a readout from the first plate, and 15 minutes for each plate thereafter.
It should be noted that Formulatrix will also offer a multiple-module system for customers who require "ultra-high throughput," Rawlins said. The additional modules increase throughput by addressing the rate-limiting steps of the digital PCR workflow, primarily the partitioning step. Each additional module will enable a user to generate 10 plates per hour, significantly increasing throughput, but also taking up more bench space.
In addition, while the previous Constellation system used 96-well plates, with each well containing 498 partitions, the next-gen system will have a choice of 96-well plates with 8,000 partitions per well or 24-well plates with 36,000 partitions per well. The latter configuration "offers a really high dynamic range for rare target amplifications, and will make us really competitive in that market," Ventura said.
Constellation's nearest competitor, the QuantStudio 3D system from Thermo Fisher Scientific, requires users to engage in multiple manual steps on three instruments, as do droplet-based digital PCR platforms, such as Bio-Rad's QX200 platform. Despite these manual steps, the QuantStudio workflow can perform thermal cycling on up to 24 chips at a time, and takes less than 30 seconds to read each chip.
The QuantStudio 3D nanofluidic chip can also partition samples into as many as 20,000 independent reaction wells, putting it in the same ballpark as the Constellation. Bio-Rad's system also fractionates samples into 20,000 individual reaction volumes, while RainDance's digital PCR technology, which is now owned by Bio-Rad, can partition samples into millions of droplets.
Generally speaking, a higher number of individual reaction volumes in digital PCR yields greater sensitivity and ability to detect rare events, but only to a certain extent — most researchers have found that something in the tens of thousands is adequate for most digital PCR experiments. Droplet-based systems also have a much more complicated workflow than plate-based systems, and are more expensive.
The other feature that sets the Constellation apart from other digital PCR platforms on the market is five-color multiplexing, which theoretically will allow users to assay for five different targets simultaneously. Other digital PCR platforms can only assay for two targets at a time, though researchers have developed workflow modifications to get that number up to four targets. It remains to be seen how important it will be to be able to assay for five targets in parallel, but as digital PCR becomes more mainstream, researchers are sure to desire an increased number of targets per assay, just as they have with qPCR.
Whereas Formulatrix previously was targeting a price of about $50,000 for the original Constellation instrument, Ventura said that the next-generation platform has a list price of about $90,000, not including additional modules. This is about double the price of a QuantStudio 3D platform, and similar to the cost of a Bio-Rad QX200 system. Meanwhile, Formulatrix quotes a "per-sample" cost for consumables. Depending on the plate selected, users may end up paying between $1.50 and $2.50 per sample, which the company thinks will also differentiate it from its competitors.
"Looking at Bio-Rad and Life Tech, the consumables rate should be less, the run rates should be less, and where we see one of the biggest game changers is that both those companies have proprietary reagents for the instrument to operate," Ventura said.
"We are not a reagent manufacturer, [though] down the road that may change through different channel partners, but at the moment, we are validating a number of suppliers of various master mixes," he added. "Our goal is to be reagent agnostic to allow someone to use their existing qPCR assays and convert them to a digital [assay] without having to read out a completely new chemistry from a new supplier."
An early beta tester — in fact, the earliest user and almost more of a collaborator, according to both companies — is pharmaceutical firm Regeneron, which has been running the new Constellation system in its DNA core facility for several months.
Researchers there echoed many of the same advantages stated by Formulatrix.
"We introduced digital PCR as something we offer to the company about two or three years ago," said David D'Ambrosio, a research associate at Regeneron. "Since then, it's been growing almost exponentially."
With the Constellation, D'Ambrosio said, the core lab has gone from doing about 96 assays per day to about 384 multiplexed reactions every three to four hours by running four 96-well plates simultaneously, with each plate containing multiplexed assays.
"We're a very high-throughput lab so we have a lot of automation," D'Ambrosio said. "The system lends itself well to automation just because of the way the plates are set up, and the way that it primes itself. So this represents a big step for us. We can go from processing small and really medium throughput to all of a sudden being able to support a very large throughput."
Rostislav Chernomorsky, director of automation technologies in Regeneron's DNA Core facility, added that automation is also a huge time saver. "All the technologies on the market today are very laborious and intensive, with a lot of hands-on time. This frees us to do many other things in the lab, as everything is built in and allows us to do many other things at the same time."
D'Ambrosio admitted that the initial investment in the platform is a little higher than competing platforms, but, he added, "I think per sample, if we were running at full capacity, we'd be paying less."
Like many digital PCR users, Regeneron plans to primarily use the platform for copy number analysis and low-frequency allele detection, especially in liquid biopsies. "Sometimes we're looking for 100 or fewer cells in 500 microliters of blood, and the digital lends itself very well to that," D'Ambrosio said. "I think right now, we can pick out [about] 0.1 percent allele frequencies."
Finally, Regeneron, through its relatively new Regeneron Genetics Center, conducts a lot of next-generation sequencing — perhaps much more so than an average pharmaceutical company of its size. In 2014, it partnered with Geisinger Health System of Pennsylvania to study the genomes of 100,000 patients to improve drug development and patient care.
Earlier this year the center announced a new research alliance with GlaxoSmithKline and the UK Biobank to sequence the genome of each of the biobank's 500,000 volunteers. A spokesperson said this week that as of this month the center has sequenced DNA from more than 200,000 individuals and is currently sequencing at a rate of more than 200,000 samples per year.
A high-throughput digital PCR system is crucial to this work because it allows Regeneron's researchers to quickly and easily validate NGS results, said D'Ambrosio and Chernomorsky.
"If they have a minor hit, we can look back with digital at the probe of choice and are able to validate it with a much higher level of accuracy, without having to do that extra deep sequencing," D'Ambrosio said.
"Once we do either … sequencing or gene expression analysis … this can help us understand in much more detail what exactly is happening on a molecular level, because we are able to detect much smaller changes than potentially with any technology out there," Chernomorsky added.