This article has been updated to clarify details of the research agreement between CHOP and Omixon.
NEW YORK – As nanopore sequencing continues to advance into the clinic, next-generation sequencing assay makers are starting to harness the technology to develop transplant diagnostic products that promise to have a fast turnaround and are cost-effective.
Last month, Hungarian molecular diagnostics firm Omixon released a new single-locus human leukocyte antigen (HLA) typing kit called NanoType Mono that uses nanopore sequencing. Meanwhile, Dutch company GenDx, which is part of Eurobio Scientific, launched NGS-Turbo, an assay also designed for high-resolution HLA typing using Oxford Nanopore Technologies platforms.
While the new assays are currently for research use only (RUO), both Omixon and GenDx said they are working to obtain regulatory approval for their clinical use under the EU’s In Vitro Diagnostic Regulation (IVDR).
Encoding proteins that play a significant role in the immune response, the HLA system "is the most diverse set of genes in the human genomes," said Omixon Founder and CEO Attila Berces. "It has a very diverse set of polymorphisms, and it has specific challenges, both for the wet lab as well as for the bioinformatics."
Previously, the company developed short-read sequencing-based HLA typing products using the Illumina platform, based on technologies licensed from Children's Hospital of Philadelphia. While Illumina sequencing has improved the resolution of HLA typing in recent years compared with other molecular techniques, such as Sanger sequencing and PCR, turnaround time and cost still remain a challenge for certain users, Berces noted.
To that end, nanopore sequencing has "tremendous benefits" for HLA typing, said Omixon VP of Product Innovation Gregory Werner. In addition to low upfront instrument costs, the long reads allow for HLA genes to be sequenced in full, leading to better phasing and typing with higher resolution.
Moreover, the real-time sequencing capability of nanopore sequencing can lead to a faster turnaround time of just hours, Werner said, which can be crucial for rapid HLA typing of organ donors in time-sensitive cases. In contrast, an HLA typing test on an Illumina platform typically runs more than a day, he noted.
Omixon first launched its nanopore sequencing-based HLA typing assay in September of 2022. Named NanoType, the kit is compatible with blood DNA samples and can amplify all 11 HLA loci — HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 — in a single tube using long-range multiplex PCR before nanopore sequencing.
According to Omixon’s website, customers using NanoType have the option to run up to 12 samples at a time. In that case, the turnaround time, from sample to result, is 16 hours, including less than five hours on the nanopore sequencer and about two hours of hands-on sample prep time.
Alternatively, users can use NanoType for single-sample urgent typing, which promises to deliver results in about five hours, including less than four hours for sequencing and a 50-minute hands-on prep time.
The difference between NanoType and the more recently launched NanoType Mono products is that the latter allows labs to perform HLA typing on an individual gene locus as needed, Berces said. The starter kit of this product line, dubbed NanoType Monoall, includes separate tubes for each of the 11 HLA loci and is configured to accommodate 24 samples. Meanwhile, customers can also purchase NanoType Mono kits, configured for 24 or 96 samples, for individual gene families.
According to Omixon, when running 24 samples on the HLA-B NanoType Mono kit, the turnaround time from sample to results is about six hours, including an hour of hands-on time. Additionally, Werner said, the long-read data is analyzed using the company’s proprietary NanoTyper software, which specifically caters to nanopore-based typing.
Given the RUO status of the company’s kits, they are currently only marketed for research applications. However, Werner noted that the company has submitted an application for the NanoType kit in July for clinical clearance in the EU and is "on the fast track" with the notified body to obtain CE-IVD marking under the IVDR.
The long-term goal of the company is to transition from the Illumina sequencing-based kit, which is named Holotype HLA and has been CE-IVD marked in Europe, to the nanopore sequencing products, Berces said.
Additionally, clinical labs in the US have the possibility to develop and validate their own laboratory-developed tests (LDTs) using the NanoType kits, he said. Omixon currently has no plans to seek FDA approval, he noted, though it is "always thinking about it."
"Proper characterization of the [HLA] genes is challenging, and next-generation sequencing platforms have proven particularly helpful to this challenge," said Dimitri Monos, director of the immunogenetics laboratory at CHOP whose lab developed the technologies that were licensed to Omixon. "Unless you have an accurate and thorough characterization of the genes, you cannot define with precision what is the allele you have in your patients or in your donors."
Monos said his team started to develop NGS assays in the early 2010s and subsequently licensed a subset of primers for Illumina sequencing-based HLA typing to Omixon in 2014.
Recognizing some of the inherent limitations of short-read NGS, the group later started to explore the utility of nanopore sequencing in HLA typing, he said. In 2020, he and collaborators published a proof-of-concept study demonstrating the ability of nanopore sequencingto characterize 11 HLA loci rapidly and comprehensively.
According to Monos, Omixon’s most recent licensing agreement with CHOP covers additional primers for more HLA genes, as well as the workflow that enables HLA typing using the Oxford Nanopore platforms. In addition, the team has signed a collaborative research agreement with Omixon to further develop nanopore-based HLA typing tests.
Despite the ongoing partnership with Omixon, Monos said the company’s product is not exactly the same as what is currently used by his team, and the lab is not obligated to use Omixon’s products. As part of the research agreement between CHOP and Omixon, the collaborators are hoping to develop a kit that satisfies his lab’s requirements, Monos said, which will be available for Omixon to license and commercialize.
Two of the areas that his team is continuing to improve, for instance, are speed and turnaround time. In the case of a newly-deceased donor, the optimal time to complete the typing is less than five or six hours in order to preserve the integrity of the organ, Monos pointed out.
Omixon is also not the only company betting on nanopore sequencing for HLA typing in the market. Shortly after the company launched the NanoType Mono products, Dutch diagnostic firm GenDx released a nanopore-base HLA typing kit named NGS-Turbo. It remains to be seen how the two companies’ products will compete with each other moving forward.
Similar to Omixon, GenDx also previously developed an Illumina sequencing-based HLA test, named NGSgo, which features multiplexed HLA amplification of 11 loci for sequencing and has already been approved for clinical use under IVDR.
According to GenDx Chief Marketing Officer Hanneke Merkens, one of the main selling points for the company’s nanopore kit is speed. "Twenty-four hours is the absolute bare minimum you need with the Illumina kit," Merkens said. "Normally, the turnaround time is one and a half days to two days." In contrast, NGS-Turbo promises to achieve high-resolution HLA typing in as little as three hours, from blood sample to result.
NGS-Turbo is designed to work with Oxford Nanopore’s MinIon and GridIon sequencers and includes two workflow components: NGS-TurboAmp and NGS-TurboPrep. Specifically, NGS-TurboAmp can achieve multiplexed rapid amplification of all 11 HLA loci within 90 minutes.
After that, users can run NGS-TurboPrep, a single-tube library preparation process that can be performed at room temperature in 30 minutes and is designed "as dummy-proof as possible," Merkens said.
Nanopore sequencing takes from 20 minutes to 60 minutes, and the data are analyzed directly with GenDx’s proprietary NGSengine-Turbo, which is designed for analyzing Oxford Nanopore long reads.
Similar to Omixon’s products, NGS-Turbo is also currently marketed as an RUO assay. While IVDR compliance is "definitely the route" the company is taking for the product, Merkens said, FDA approval is "not on the agenda yet," given the possibility of developing the kits into LDTs.
At the moment, NGS-Turbo is designed to analyze one sample at a time. The next version of the product will allow customers to type one to 96 samples in one go, Merkens said.