NEW YORK – Molecular diagnostics company Asuragen is commercializing a targeted nanopore sequencing assay that promises to analyze multiple genes implicated in high-prevalence carrier screening conditions in a consolidated workflow.
The Austin, Texas-based firm, a brand of Bio-Techne, is slated to launch the test for research use only (RUO) this fiscal year but is staying mum on the exact timeline as well as on plans to further develop the assay into a clinical product.
"We decided to pursue consolidation of genes that are otherwise challenging or intractable by short-read sequencing, using the long-read sequencing platform from Oxford Nanopore," said Asuragen CSO Brian Haynes. "What is fantastic about nanopore sequencing and our approach is that it cannot only address the content that is really challenging by short reads, but it can do everything else that short-read sequencing is already doing."
Dubbed the AmplideX Nanopore Carrier Plus kit, the product, developed in partnership with Oxford Nanopore Technologies, contains all necessary reagents for analyzing 11 genes associated with nine common carrier screening conditions, Haynes said. The assay utilizes short- and long-range PCR to enrich for these target genes, which tend to be difficult to examine by short-read sequencing due to tandem repeats, copy number variations, and structural variations.
AmplideX Nanopore Carrier Plus is compatible with several sample types, including whole blood, cheek swabs, and saliva samples, according to Asuragen’s website. After nucleic acid extraction, 40 ng to 100 ng of DNA is used for the PCR reactions.
Haynes said the company has distributed the 11 genes over four PCR mixes, giving users the flexibility to combine their desired gene targets in one experiment. The first PCR mix targets CFTR, a gene responsible for cystic fibrosis, as well as SMN1/2, which are linked to spinal muscular atrophy (SMA). The second mix aims for FMR1, mutations in which can lead to fragile X syndrome. The third mix looks at HBA1/HBA 2 as well as HBB, which are responsible for alpha thalassemia and sickle cell disease, respectively. The final PCR mix interrogates gene inversions in F8, which are associated with hemophilia A; GBA, which is linked to Gaucher disease; and CYP21A2/TNXB, which are implicated in 21-hydroxylase deficient congenital adrenal hyperplasia (CAH) and Ehlers-Danlos syndrome (EDS).
Each mix undergoes a two-stage PCR reaction, Haynes said, and the amplicons can subsequently be pooled for sequencing and analysis. The company also harmonized the PCR conditions for the first three mixes, enabling their reactions to be carried out in the same thermal cycler.
Haynes said the AmplideX Nanopore Carrier Plus assay is optimized for the Oxford Nanopore GridIon platform using R10.4.1 flow cells and chemistry. The workflow allows for flexible batching of 12 to 96 samples per flow cell. The estimated hands-on time for a 24-sample batch, for instance, is four hours, he noted.
In addition to the reagent kit, the company will also launch a companion analysis software for data analysis and reporting. Named the AmplideX Reporter, the software will take in nanopore sequencing data to help conduct automated summary QC and variant calls, Haynes said.
Currently, the sample-to-report turnaround time for the assay, if all 11 genes are analyzed, is about three days. Its list price is still being determined, he added.
To demonstrate the technology’s feasibility and utility for analyzing SMN1 and SMN2 variants, Asuragen has collaborated with researchers at Al Jalila Children’s Specialty Hospital in the United Arab Emirates.
In a preprint posted on MedRxiv earlier this year, researchers from Al Jalila Children’s and their collaborators described testing a prototype of Asuragen’s assay for SMN1 and SMN2 profiling on 750 samples, including presumed normal samples and patients with known SMN1 and SMN2 genotypes.
The study, which has not been peer-reviewed, concluded that the workflow could detect SNVs, indels, and CNVs with more than 98 percent accuracy across all samples. The authors also noted that the turnaround time for the assay is less than 48 hours for a 24-sample batch, using an Oxford Nanopore Mk1B flow cell connected to a computer with a recommended GPU.
"We really wanted to have a comprehensive assay that [for instance] can do copy number variants, any rearrangements, but at the same time being cost-effective," said Ahmad Abou Tayoun, director of the Genomics Center of Excellence at Al Jalila Children’s Hospital and the corresponding author of the study. Given the high burden of recessive diseases, such as SMA, in the region due to consanguinity, Tayoun said, carrier and premarital screening can be a powerful tool for the Middle Eastern populations to combat these diseases. He also noted that for screening applications, throughput, accuracy, and cost are important attributes of a test.
Tayoun’s team estimated that the per-sample material costs for the SMN1/2 assay were less than $20, excluding the PCR enrichment costs, which were still not finalized. The calculation also did not include instrument costs, but Tayoun noted that one potential advantage of nanopore sequencing is the sequencer’s accessibility and lower upfront investment.
In addition to the study from Al Jalila Children’s Hospital, Haynes said Asuragen has also partnered with external collaborators from other institutions to further evaluate the company’s product in "many different geographies."
With its assay, Asuragen is eyeing an increasingly crowded carrier screening market, where many NGS-based assays have already been commercialized. Natera’s Horizon genetic carrier screening test, Myriad Genetics’ Foresight carrier screening test, and Quest Diagnostics’ QHerit expanded carrier screening test, for example, all tap sequencing to help screen for a slew of conditions including cystic fibrosis, spinal muscular atrophy, fragile X syndrome, and sickle cell anemia.
Haynes said one reason the company bets on nanopore sequencing is the technology’s flexibility and accessibility. Moreover, he pointed out that it can sequence molecules of all sizes and enable comprehensive analysis of the target genes.
With the Nanopore Carrier Plus kit still undergoing commercialization, it remains to be seen how the product will perform in the real world and how it will stack up against competing offers.
Haynes also declined to comment on if or when Asuragen plans to validate the AmplideX Nanopore Carrier Plus assay for clinical use. "We are definitely looking at a regulatory strategy across our entire [product] portfolio," he said.