NEW YORK – Paragon Genomics plans to launch an updated version of its CleanPlex SARS-CoV-2 next generation sequencing (NGS) panel to enable high-throughput COVID-19 clinical research, diagnostic testing, and surveillance.
The Hayward, California-based firm believes that customers will be able to use the panel to simultaneously sequence over 20,000 samples. The company expects to offer the updated kit within the next three to four months for research-use-only purposes before applying for Emergency Use Authorization from the US Food and Drug Administration.
Paragon's current SARS-CoV-2 NGS kit, launched in March, applies the firm's CleanPlex multiplex PCR technology to analyze the entire genome of the virus. The firm currently offers the panel for research and surveillance purposes.
Paragon Genomics CEO Tao Chen explained that in the current CleanPlex protocol a user extracts RNA from a patient sample containing the virus, which can include nasopharyngeal swabs or an infected tissue. The firm recommends about 50 ng of purified total RNA, incorporating both human and viral RNA.
After amplifying the RNA in a multiplex reverse transcription PCR reaction, Paragon's kit then biochemically removes primer-dimers, non-specific PCR products, and complex molecular debris in a proprietary digestion reaction followed by magnetic bead purification. The panel then uses PCR primers to tag remaining capture sequences and barcode sample indexes in a PCR indexing reaction.
Dawn Casey, director of business operations at Paragon, noted that the process can amplify the entire genome of the virus from RNA to sequence-ready libraries in less than six hours.
Customers can then process up to 2,688 patient samples in a single run using sequencing instruments from companies including Illumina, MGI, and Thermo Fisher Scientific.
Casey explained that Paragon's new SARS-CoV-2 panel will perform in essentially the same manner as the currently available assay. However, the kit will also include additional barcoding steps at the end of the four-step process
The high-throughput version of the panel will allow customers to simultaneously sequence over 20,000 samples in a single run.
Chen argued that Paragon's NGS assay stands out from RT-PCR based methods for COVID-19 detection because it offers a higher sensitivity and accuracy, which he believes will translate to stronger clinical results and effective treatment. The panel will also detect as low as a single viral copy in a patient sample.
"Because we're targeting the entire genome, as opposed to a handful of amplicons like RT-PCR, we're preferentially amplifying the SARS-CoV-2 virus over other viruses or other contaminants that are present along in the sample," Casey added. "We're able to detect the single viral copy as long as we can amplify it."
Based on initially limited data from Paragon's customers, Chen said that the firm has seen high specificity for the current assay, or "probably over 99 percent."
Because Paragon is currently filing patents for the assay, Chen declined to discuss further details about the additional steps the new panel will include.
Chen argued that the current CleanPlex SARS-CoV-2 Panel reduces sequencing costs by 99 percent when compared to hybrid capture and metagenomics sequencing methods. Based on product specifications from competing methods, he explained that they usually require 8 to 10 million sequencing reads to "comfortably cover" over 99 percent of the viral genome.
In contrast, Paragon's amplicon-based SARS-CoV-2 panel only requires 50,000 to 100,000 reads to obtain the same or better results.
Casey said that the new panel will drive down the price of NGS testing because using 10,000 to 20,000 samples per run will reduce the amount of reagents needed, compared to several runs of 2,000 samples at a time.
However, Chen acknowledged that the Paragon will need to validate the new panel on multiple COVID-19 samples in order to further prove its limit of detection. The firm has partnered with an undisclosed sponsor lab to test the new panel on COVID-19 samples.
While the firm is still determining the price of the updated kit, Chen believes that if a customer can pool up to 20,000 samples in a single NovaSeq run, they can reduce the cost of sequencing to about $3.60 per sample.
Chen said that Paragon is also currently working on variations of the method for high-throughput biomarker discovery, cancer detection, and population screening.
Several academic laboratories are attempting to use viral genome sequencing assays from firms like Paragon to complement existing RT-PCR assays for research, diagnostic, and surveilling purposes.
Xiaowu Gai, director of bioinformatics at the University of South California's Center for Personalized Medicine, and his colleagues initially sought to analyze SARS-CoV-2 genomes when the virus hit the US earlier this year. His team has used Paragon's NGS assay to help determine how to treat and minimize the transmission of the disease.
"Our task was to evaluate diagnostic tests for COVID-19, and [thus] we started validating FDA EUA RT-PCR assays," Gai said. "While Paragon's NGS assay had only been tested on two to three clinical samples at the time, we were surprised the test worked very well from the get-go [in our lab]."
Gai noted that his team has been applying both Paragon's and Thermo Fisher Scientific's Ion AmpliSeq SARS-CoV-2 panels in their lab to develop a research-use assay and analyze genetic data from the virus. After performing a full side-by-side comparison on COVID-19 samples, the researchers found that both assays performed the job effectively.
However, Gai highlighted Paragon's technology did not require much tinkering to successfully detect COVID-19 in the initial clinical samples.
"Paragon's assay is also robust in that it works on a wide range — from six to seven magnitudes — of viral loads in a sample," Gai said. "It's [also] not that expensive of an assay relative to other NGS [methods], but it is expensive compared to RT-PCR assays to detect the SARS-CoV-2 virus in patients."
Gai argued that the Paragon could improve the existing panel by increasing the sample throughput while minimizing the overall costs of the assay. If the firm dropped the cost of the assay "even further," he believes the panel could become an ideal choice for an NGS-based COVID-19 assay. However, he noted that increasing the sample throughput per sequencing run can often lead to challenges when analyzing the sequencing results.
"After [receiving] the data, you have to ask how to make sense of it, since you're trying to come up with very advanced results and take advantage of them for clinical applications," Gai said. His team has spent a huge amount of effort to minimize background noise and other issues related to sequencing an increased number of samples.
While Paragon will submit an EUA application for the current SARS-CoV-2 panel, Chen hopes to eventually achieve FDA 510(k) clearance in the US, as well as a CE mark in Europe. The firm therefore anticipates launching further validation studies later this year to demonstrate the current test's clinical sensitivity and specificity on larger cohorts of patients.
After completing development of the updated version, Paragon will release the panel as a research-use-only product before pursuing an FDA EUA. CHen noted that the current panel and future higher throughput version will need separate FDA EUAs due to slightly different protocols.
In addition to collaborating with researchers on SARS-CoV-2 genomic research and diagnostic testing, Paragon believes the updated test could be used for disease surveillance. Chen argued that collecting sequencing information will help determine the history of the spread and measure the breadth of the virus.
"In order to track a virus's path, researchers need to have a full sequence of the genome to determine how it's mutating, where it's mutating, and what the mutation rate is," Casey added. "Because our assay targets the full genome, we're able to get the full information about the virus."
Depending on how the coronavirus and its subtypes mutate, Gai said that sequencing the entire viral genome will become critical to help devise treatments based on the severity of the disease in different patient populations.
"People are underestimating how fast this virus can accumulate mutations and spread out," Gai highlighted. "As we analyze whole-genome sequencing data on the virus across the world [in different patient samples], there are some patterns as to how new strains could eventually lead to a higher pathogenicity."
Acknowledging that Paragon unexpectedly expanded its infectious disease research and offerings with the first "ready-to-use" SARS-CoV-2 panel, Casey argued that the firm still expects to widen its oncology and genetic disorder pathways. However, she emphasized that Paragon was prepared for the pandemic since it had been developing infectious disease panels through custom panel offerings.
"It's a normal part of our business, which is part of the reason we've been able to adapt and provide an assay so quickly," Casey said. "The custom panel allows us to work on several applications … including infectious diseases."
In the long term, Paragon aims to enter the in vitro diagnostic space with some of its existing oncology and infectious disease panels. To do so, the firm will partner with additional commercial organizations and medical institutions. In addition, the firm may also develop an even higher throughput SARS-CoV-2 assay.
"Another higher throughput version could allow the pooling of 49,000 samples, which will further reduce the sequencing cost to $1.50 per sample," Chen explained. "[However,] we will consider developing the 49,000-sample version if there is demand [for it] in the future."