May 05, 2015 | Tony Fong
NEW YORK (GenomeWeb) – Aiming to simplify and improve the targeted sequencing workflow, LC Sciences（联川生物美国总公司） is building out its VariantPro multiplexing PCR technology with an eye on a fourth quarter launch.
The company is in the midst of testing the technology and recently sent out a call for beta testers to, among other things, generate test datasets for the technology. If all goes as planned, LC Sciences anticipates making VariantPro available as part of its service offerings in the fourth quarter, followed shortly afterward by a launch of the technology as a kit for researchers to run in their own laboratories. If successful, it would make the technology the second commercially available product from the Houston-based biotechnology firm.
VariantPro is a multiplex PCR-based technology designed to overcome issues that have plaguedother PCR-based methods, such as low uniformity, sequence dropout, a complicated workflow, lengthy hands-on time, and high upfront equipment costs, according to LC Sciences.
"When you do conventional multiplexing PCR, you need to do a lot of optimization," LC Sciences Co-founder and Chief Technology Officer Xiaochuan Zhou told GenomeWeb last week. "In order to overcome [those issues], we designed a process in such a way that a lot of things can be done by [automation]."
For targeted sequencing, typically two to three steps are needed at the front end — multiplex PCR followed by ligation, and then followed by another round of PCR, he said. With the VariantPro technology, that can achieved in one step.
Underlying the technology are two innovations, Zhou said. The first, dubbed Relay-PCR, is a multiplex PCR method that allows a researcher to target specific primers, including the index library primer, and the polymerase in one tube. One pair of common primers and multiple pairs of specific primers are mixed with a genomic DNA sample in one tube, and two functionally separated reaction phases — target replication and library amplification — are combined in one PCR run, resulting in a simplified workflow, LC Sciences said.
The innovation of Relay-PCR is that it limits the role of specific primers to target selection in the first two thermal cycles, enabling an automatic switch to common primers in the remaining thermal cycles for library amplification.
"From the third cycle on until the end of [the] cycles, [the process involves] amplification," Zhou said. Eliminated is the need to perform multiplex PCR, then purification, followed by ligation, and then more PCR, he added.
Chris Hebel, LC Sciences' vice president of business development, told GenomeWeb that VariantPro does away with primer-specific variation in the amplification process. "You've got tens, hundreds, thousands of different primer pairs that have different thermodynamic efficiencies, so some amplicons are going to be overrepresented" compared to others.
A researcher can eliminate the primer-specific variation by selecting targets with specific primers in only the first few cycles, "but if you then amplify targets using those specific primers … you'd have that variation exponentially amplified."
With VariantPro, though, a researcher can capture the amplicons in the first couple of cycles, and starting with the third cycle, the scientist can begin the common primer amplification, "which is just a single PCR reaction. There's no variation due to differences in primers," Hebel said.
With VariantPro, the two steps are contained within the same PCR reaction "based on the design of the primers, the temperature, [and] the concentration of the primers in the reaction," he said. "That has enabled this automatic switch."
The second innovation of VariantPro is the Omega-Primer, made up of three segments —a five-prime arm, a three-prime arm, and a separation segment between the two arms. The five-prime arm ensures stable binding to a corresponding template, and the three-prime arm checks sequence specificity and initiates polymerase extension.
According to LC Sciences, the use of the separate binding sections "provides primer design freedoms that balance priming specificity and binding strength." Omega-Primers have shorter three-prime arms than that of regular primers, and so have a statistically lower probability of forming amplifiable primer-primer dimers in a multiplex PCR setting.
Also, because only the short three-prime arms are incorporated into amplicon products, the sections of native templates in sequencing reads are maximized, the company said.
"Because you have the two binding sections of this primer, it gives you additional freedom in terms of designing primers for any difficult-to-target region, so there are going to be target regions that others have tried and failed to design targets to that we will be able to target," Hebel said. He acknowledged, however, that because VariantPro has not been used in real-world applications, this is theoretical.
Because VariantPro can target more than 20,000 targets in one reaction, it is particularly suited for larger targeted sequencing projects and custom sequencing projects, he said. Zhou added that the technology "can be used beyond our current kit development for any method like a pathogen panel or anything involving multiplexing PCR."
LC Sciences has operated as a service firm since its inception, and to date the only product it makes available to outside researchers is OligoMix, customized synthesized oligos for multiplexing reactions. OligoMix has been on the market for about a decade, and for the past several years, as target capture and multiplex PCR of large numbers of targets has become increasingly popular, LC Sciences saw that many of its OligoMix customers were using it to develop its own target capture methods. It decided "that it makes sense for us to develop our own technology for the use of this oligo mix for targeted sequencing," Hebel said.
As LC Sciences builds out VariantPro, which is platform-agnostic, the firm is looking to generate more data and provide actual case data. As part of that effort, the company put out a call last month for beta testers for the technology with the goal of testing the VariantPro's limits.
Additionally, Hebel said that researchers may have content, such as panels of genes, that they want to target for specific diseases or pathways, but for which current technology is not able to deliver a panel or products. LC Sciences wants to collaborate with such researchers who have the biological content and develop with them clinically relevant panels of targets.
The company plans to launch VariantPro in the fourth quarter as part of its service offerings, and then as a kit, as incorporating it into its service business is a "more straightforward route" for commercialization than launching it as a kit, Hebel said.
LC Sciences has not determined a price for VariantPro yet, but "we imagine that we can get to below $100 per sample for targeting, let's say thousands of targets," Hebel said, adding the average price currently with competing technologies is around $200 per sample.
The firm also is "considering strongly" developing assays using VariantPro, Hebel said. "I wouldn't say it's in the plan book right now … It's more likely that our technology will be adopted by others who have panels," he said.