SAN FRANCISCO (GenomeWeb) – The Guy's and St. Thomas' NHS Foundation Trust has developed a nanopore sequencing-based test for Huntington's disease, laying the foundation for future nanopore sequencing-based clinical tests. The test will not initially be used as a first-line test, but as a reflex test in cases where results from the standard PCR-based test are not clear.
Researchers from Guy's and St. Thomas' NHS Foundation Trust collaborated with Viapath, King's College London, and the London South Genomic Laboratory Hub to develop the test, and they plan to publish on the validation of the test in a peer-reviewed journal. In addition, the test has been accredited for clinical use by the UK Accreditation Service, which provides accreditation for pathology laboratories under ISO15189.
Deborah Ruddy, a clinical geneticist at Guy's and St. Thomas's, said that the group who developed the Huntington's test has a particular focus on "translating new technologies into the diagnostic sphere." Getting the Huntington's test on Oxford Nanopore Technologies' MinIon accredited for clinical use also opens the door to develop other clinical tests on the MinIon, she said, and the team next plans to focus on developing tests in the oncology and infectious disease spaces for applications that take advantage of the long reads.
Graham Taylor, director of genomics innovation at Viapath and a professor at Kings College London, said the researchers developed and validated the Huntington's targeted sequencing-based test on 100 samples. The test targets the HTT gene, which contains a tri-nucleotide repeat. Normal individuals have fewer repeats — typically up to only around 35 — while individuals with Huntington's will have 36 or more repeats, and can have up to more than 100. In addition, individuals with a repeat size between 30 and 35 do not typically develop the disease but risk passing it on to children.
The test uses PCR primers to target the HTT gene, which is then sequenced on the MinIon. Diagnosis of Huntington's is based on counting the number of repeats, so single-base accuracy is not critical, Taylor said.
The current standard Huntington's disease diagnostics are fluorescent PCR assays — HD1-HD3 and HD2-HD5. These assays measure the number of repeats on each allele of the HTT gene. The team validated the nanopore assay to detect alleles greater than 50 repeats and said that the nanopore assay would initially be used in cases where the PCR assay is homozygous for both alleles to ensure that a large expansion was not missed.
In addition, Taylor noted that the research team is continuing to revise the algorithm to improve performance.
The standard PCR assay is accurate, "but doesn't give information about structure within the repeat," Taylor said. Understanding the structural nature of the repetitive region does not currently impact diagnosis, Taylor said, but there is research into the impact of interruptions within the repeat expansion as well as whether and how the repeat expansion changes over time, which sequencing could be better positioned to address.
Taylor noted that the goal is to have a turnaround time for the clinical test of a few days. He declined to disclose the cost of the assay, but said it was comparable to current PCR diagnostic tests for Huntington's disease. He said that the team is continuing to work on reducing costs and plans to evaluate Oxford Nanopore's Flongle device, which could help bring costs down. In addition, he said, the team is evaluating using the Cas9 enzyme, rather than PCR primers, to target the HTT gene region.
Over the last several years, there has been growing interest in using next-generation sequencing to diagnose repeat expansion disorders.
Researchers from the Parkinson's Institute and Clinical Center, for instance, developed a targeted sequencing test using Pacific Biosciences' long-read sequencing technology to identify repeat expansions in the ATXN10 gene, which can cause spinocerebellar ataxia.
And while short-read technology still struggles to accurately read through repetitive regions, researchers, including from Illumina and the Walter and Eliza Hall Institute of Medical Research in Melbourne, have made progress in developing bioinformatics tools that can make short-read sequencing feasible.
Going forward, the UK team intends to develop other clinical tests using the MinIon, including for oncology and infectious diseases. For oncology, Ruddy said the group plans to focus on chromosomal translocations involved in cancer.
For infectious disease testing, Jonathan Edgework, medical director at Viapath, said that researchers are evaluating the MinIon for 16S sequencing and metagenomic sequencing.
For 16S sequencing, the goal is to use the MinIon to quickly analyze a clinical sample "where you just want to know what bacteria is there," he said. Such a test would be designed to replace 16S sequencing that is currently done on Illumina sequencing platforms and takes between four and five days to get a result. Such a test on the MinIon could be done within 24 hours, he said.
A metagenomic sequencing test would come after a 16S test has been developed and shown to be robust, Edgework said. For that, he said, the team is interested in evaluating respiratory infection samples. A rapid metagenomic sequencing test in such cases could help the infection control team make decisions about how to manage a patient, he said, including deciding whether the pathogen is of potential pandemic significance, thus requiring a patient be isolated.
"If you have to wait a few days or weeks before you know what you're dealing with, that becomes a significant challenge, and some of these pathogens can be fatal within that time frame," he said.