NEW YORK – Paragon Genomics has developed new target enrichment assays that use its CleanPlex multiplex PCR-based technology, as well as a new technology for gene fusion detection.
The Hayward, California-based company recently launched a cystic fibrosis targeted resequencing assay. It also released the results of collaborations with Admera Health and RareCyte using custom target enrichment kits.
Paragon's flagship CleanPlex target enrichment solution involves a multiplex PCR technology that allows researchers to multiplex more than 20,000 amplicons in a single reaction. According to Dawn Casey, director of business operations at Paragon, the method typically produces sequencing libraries within two to six hours and works with downstream instruments such as Illumina's MiSeq, Thermo Fisher Scientific's Ion Torrent, and MGI's sequencing platforms.
Launched earlier this month at the 2019 Association of Molecular Pathology Conference in Baltimore, Maryland, Paragon's new CleanPlex Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Panel evaluates somatic and germline variants across the CFTR gene. According to Casey, Paragon selected cystic fibrosis because of customer requests.
While the CFTR panel normally uses between 1 and 10 ng of DNA, Casey said it can also run on lower and higher amounts of genomic DNA isolated from buccal or blood cells.
"Up until now, people have been biased against amplicon methods because of the background noise, the primer dimers, non-specific reads, and nonuniformity," Casey explained. "But because of our background [reduction] technology and proprietary design algorithm, we've been able to eliminate those issues."
Paragon also said it will soon begin an early-access program for its CleanPlex Fusion Detection Method, which looks for both known and novel gene fusions and uses a different background cleaning method than the other CleanPlex assays.
Paragon CEO and Cofounder Tao Chen explained that the method involves a template-switching step at the beginning to reverse-transcribe RNA to cDNA, where a universal sequence is added to the 3' end of the cDNA. For the second PCR step, one universal primer and one target-specific primer are used to amplify the target gene, allowing researchers to detect novel fusions.
However, Chen declined to disclose further details about the fusion method because the firm is currently filing related IP with the US Patent and Trademark Office.
Chen said that researchers can sign up to evaluate Paragon's first panel based on the fusion method, the Lung Cancer Fusion Panel, and that the firm aims to launch additional products with a custom fusion assay design service by the end of the year. He noted that Paragon selected lung cancer as the first application because the cancer harbors several gene fusions that have established drug treatments.
Paragon expects users that will participate in the fusion method early-access program to include hospitals, reference labs, or "anyone's that's oncology-focused," Casey said.
Paragon also offers a tumor mutational burden (TMB) panel that looks at 516 genes and 27,000 amplicons, which it launched as part of an early-access program at the American Society for Human Genetics annual meeting last month. Since the platform measures TMB, which often correlates with a patient's response to checkpoint inhibitors, using a targeted sequencing approach, researchers can run genomic DNA derived from blood or formalin-fixed paraffin-embedded tissue, Chen said.
Because the TMB panel requires additional cycling time to process the samples, Casey said that it requires six hours to generate a library for downstream sequencing.
Recent partnerships
Casey noted that South Plainfield, New Jersey-based Admera Health collaborated with Paragon in order to improve the efficiency of its PGx One Plus assay. Designed to use CleanPlex and run on the Illumina MiSeq platform, Admera's pharmacogenetics (PGx) assay detects over 200 genetic variants in 72 genes that have been associated with more than 300 drugs.
During the Paragon workshop at the AMP meeting, Admera's head of clinical services, Min Wei, explained that Paragon partnered with the firm earlier this year to develop a customized PGx panel relying on CleanPlex. Chen noted that the panel targets 180 genes and 500 variants, including mutations that are difficult to amplify.
"We worked with [Admera] closely to provide tech support and consultation to design the novel panel," Chen said. "After … a few rounds of discussion,we delivered a high-quality, robust assay for their purposes."
During the workshop, Wei also noted that the assay had a clinical sensitivity and specificity of greater than 99 percent. In addition to offering high uniformity and a highly adaptive design algorithm, Wei said, CleanPlex's flexibility allowed her team to upgrade the number of genes that the PGx assay could detect, and her group expects to add more than 20 genes to the PGx One panel in the future.
In addition to Admera, Paragon partnered with Seattle-based RareCyte to modify the CleanPlex protocol for single cells incorporating the firm's CyteFinder platform with extremely low sample input. As part of the collaboration, RareCyte scientist Nolan Ericson applied Paragon's CleanPlex technology to identify somatic HER2 mutations in individual circulating tumor cells (CTCs) and circulating tumor DNA, which involved altering cycling parameters, primer concentrations, and clean-up changes.
After selecting cells of interest using immunostaining, Ericson and his team moved to single CTC sequencing. He noted that the process is typically difficult because a single cell only contains 6pg of DNA.
"Usually, people use whole-genome amplification to turn this into a workable amount of material," Ericson said. "But WGA has its own pitfalls, such as allele dropout [or] region dropout, which leads to false negatives and amplification bias."
Ericson's group therefore used a modified CleanPlex OncoZoom Hotspot panel to sequence individual CTCs. Launched in 2017, the assay generates a sequencing-ready library to identify over 2,900 somatic mutations across 65 oncogenes within three hours.
Using the single-cell non-WGA method, Ericson's team found that it could recover allele dropouts and minimize the number of false negatives by 60 percent and false positives by 10-fold. Ericson said that the overall workflow using CyteFinder, the CleanPlex technology, and sequencing on a MiSeq instrument could produce results within a week.
"The Paragon panels are really useful because they're very amenable to this kind of test for bypassing WGA error and doing direct targeted sequencing on the CTCs," Ericson said. "They also squeeze more targets into their panels than other commercially available panels, which is useful to us because it helps detect targets in our single tube of blood."
Ericson noted that his team plans to sequence more CTCs along with matched tumor samples in the future. He said that his group would be interested in seeing Paragon potentially finetuning its panels to allow copy number variation detection.
In addition to new products and partnerships, Paragon has also launched an online portal called "ParagonDesigner," which allows customers to design CleanPlex custom NGS panels. Researchers can use the service to evaluate the number of amplicons needed to cover their list of genes or defined genomic locations.
Chen noted that researchers can either search Paragon's database of pre-designed panels or submit custom panel design requests. Paragon scientists then build a bioinformatic file with selected amplicons and send it back to the customer, who then determine if the panel will achieve their desired target coverage rate.
After the customer files a purchase order, Paragon begins manufacturing the library kit and ships it off to the lab, which Casey said takes about two to three weeks depending on the level of amplicon customization.
Chen added that the customer can perform library prep and sequencing on an in-house platform or outsource the step to a third-party sequencing provider.
"After the initial test, clients often decide to add or remove content," Casey noted. "Not only do we help from the design side, but we also aim to reduce [design] challenges and increase the speed so that our clients can go from design to fully-optimized assays in much less time and with less hassle."
While Paragon's commercial plans for the fusion method are still in development, Chen said the firm will leverage the technology for additional downstream customer panels. He believes the technology will help Paragon work with partners that require fusion detection for cancer diagnostics.
"We plan to add more content such as fusion and [microsatellite instability] to the TMB panel, and also to partner with collaborators and customers to further validate these panels on clinical samples," Casey said. "At the same time, we can provide custom TMB panels to our customers who want to add or remove specific content from the panel."
Paragon also plans to broaden the fusion method to test solid tumor tissues and eventually develop panels for blood-based cancers. Chen envisions working with different players in the bioinformatics and automation industries — mirroring Paragon's prior partnership with Sophia Genetics — in order to streamline the firm's workflow for high-throughput NGS labs.