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WaferGen Betting that New NGS Target-Enrichment Workflow Can Help Drive SmartChip Uptake

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WaferGen Biosystems and collaborators in the laboratory of Jo Vandesompele at the University of Ghent have developed a PCR-based target enrichment workflow using WaferGen's SmartChip real-time PCR system, the company said this week.

Using the method, the group was able to achieve a high degree of sensitivity — with more than 96 percent and 97 percent of targeted regions having a coverage depth of more than 40X and 10X, respectively — as well as uniform coverage, with more than 93 percent of the 376 amplicons they targeted falling within a 10-fold difference in coverage.

WaferGen is hoping to position the new protocol as an alternative to existing single-tube target-enrichment methods, which it said often fall short of this level of coverage due to issues that may arise from multiplexing PCR reactions. The SmartChip method, in contrast, is designed to run an individual PCR reaction for each amplification reaction, thus eliminating this risk.

In particular, WaferGen officials said that this characteristic may make the SmartChip method attractive as a target-enrichment tool for CLIA labs developing their own sequencing-based clinical assays, where a high level of uniform coverage is desired; and said that an undisclosed "major US medical research institution" has already begun using the platform for this purpose.

"Our approach is geared more toward clinical applications … because CLIA labs need to get as close to 100 percent coverage as possible," WaferGen president and CEO Ivan Trifunovich told PCR Insider this week. "It's a much higher bar than research applications."

WaferGen launched its SmartChip real-time PCR system in August 2010, originally positioning the platform as a tool for highly parallel gene expression profiling studies. The system comprises a nanodispenser module for sample dispensing; consumable chips with as many as 5,184 wells loaded with target-specific primers; and the SmartChip cycler, which performs PCR thermal cycling, data collection, and amplicon melting.

However, the company has had difficulty selling the system, and has worked to find a sweet spot for the technology in the molecular biology marketplace. In December 2011, amid layoffs, the company said that it was trying to drive adoption of the platform as a tool to validate biomarkers uncovered in next-generation sequencing studies (PCR Insider, 12/1/2011); and in July of last year, it launched a program called MyDesign intended to enable customers to load their own assay content onto the SmartChip consumables as opposed to ordering pre-loaded assay content from the company (PCR Insider, 7/12/2012).

Trifunovich, who joined the company a few months before the MyDesign initiative launched, said this week that the company has seen "decent traction" in terms of SmartChip market uptake since starting the MyDesign program. However, hoping to spur additional sales, the company has continued to work on new applications for its platform, which led to the collaboration with the University of Ghent.

"The market is changing [and] next-generation sequencing obviously is playing a critical role," Trifunovich said. "Because we're doing PCR on our chip, the next logical step was to go after target enrichment, because there is a high unmet need there."

He added that WaferGen and the University of Ghent began their target-enrichment work around August, and the project moved "faster than we expected, and we are very optimistic with the results we obtained."

In a white paper published on WaferGen's website, researchers from the company and the Vandesompele lab detailed a proof-of-concept study for the new target enrichment protocol.

As part of their process, the team aimed to enrich 376 unique genomics regions across 24 samples — 16 cancer cell lines and eight control samples. They used a total of 360 picograms of DNA as input for each of the 376 unique amplicons, which were designed using the PrimerXL software pipeline from Ghent University.

The researchers spotted the amplicons onto six four-quadrant SmartChips, each containing 841 nanowells per quadrant to perform target enrichment of the 24 DNA samples — four samples per SmartChip with one sample analyzed per quadrant. They then used Bio-Rad SsoAdvanced SYBR PCR mix to perform the amplification reactions on the SmartChip cycler; purified and analyzed the PCR pools; performed Illumina Nextera XT library preparation on all 24 PCR pools; and sequenced the products on an Illumina MiSeq instrument (a single 2x150 bp run comprising 1.5Gb).

Following this workflow, 96.7 percent and 97.3 percent of the amplicons showed a coverage of greater than 40x and 10x, respectively, with 93.4 percent of the sequenced targets falling within a 10-fold coverage range, and 76.7 percent falling within a 3-fold coverage range. In addition, all known variants and mutations present in the cancer cell lines were detected using the approach.

Although the researchers did not directly compare the new SmartChip-based protocol to existing commercial target-enrichment technologies in their study, they noted that many of these technologies are single-tube, multiplex approaches in which primers, probes, or amplicons can interact with each other and result in the formation of primer-dimers and non-specific products. Further, they noted that all assays in single-tube formats compete for the same reagents, which can impact assay performance and consistency.

"What is out there right now [are] things like [Agilent] SureSelect and HaloPlex and [IonTorrent] AmpliSeq, [which are] OK for research applications, and get you anywhere between 85 to 90 percent coverage, and if you spend lots of time and also money you might get into the low 90s," Trifunovich said. "But what all these techniques are lacking is the precision of individual PCR reactions."

An additional potential advantage of the WaferGen approach, the company said, is a built-in quality control step that leverages the instrument's real-time PCR capabilities to identify irregularities during the target enrichment step prior to sample prep and sequencing.

Timothy Triche, professor of pathology and pediatrics at the University of Southern California Keck School of Medicine and pathologist-in-chief at Children's Hospital Los Angeles, told PCR Insider via email this week that these early data could go a long way toward positioning the SmartChip system as a target enrichment tool.

Triche, who is on WaferGen's scientific advisory board, said that he had long seen the potential of the platform for this application, especially now that clinical applications of NGS are increasingly focused on targeted sequencing.

"I find it appealing that the end user could generate unique targeted content other than standard commercially available offerings in order to develop custom assays for their own use, potentially as [laboratory-developed tests]," Triche said. "The particular value from my perspective is that in a world where sequencing will be available to just about every lab, most such labs … will want to sequence not only conventional protein-encoding genes, but other regions of interest that may lie in intergenic space, like promoters and areas implicated in disease states."

Triche pointed to recent evidence that perhaps as much as 70 percent of hits from genome-wide association studies lie outside coding regions of the genome. "If they are important, one might want to sequence them, as they may be regulatory regions, or at least impact normal gene function," he said. "No matter; I believe people will want to sequence regions of the genome implicated by their own research and the literature that implicates regions of interest, and the hope is that the SmartChip PCR targeted amplification will enable people to do that."

WaferGen said that the results of its study will be the subject of a soon-to-be-published peer-reviewed paper, and that the company, the University of Ghent, and other collaborators are gearing up to conduct a much broader target enrichment study.

Further, WaferGen said that a "major US medical research institution" has achieved similarly promising results for target enrichment, although Trifunovich declined to identify this collaborator.

WaferGen said that it plans to "deploy significant available resources behind the rapid refinement and commercialization" of the new workflow; and that it is "contemplated that existing system components will be complemented by a low-cost cycler and a simple dispenser that will allow the technology to be cost-effectively deployed even at smaller labs."

The company's overarching vision is that customers will see the potential of the SmartChip system both up- and downstream of NGS applications.

"You do it for your sample prep and target enrichment … and you could use it for library quantification … in one fell swoop," Trifunovich said. "Then you do your sequencing, get your results, then go back and do RT-PCR validation with gene expression or genotyping on the same platform."

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