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New Nanopore Sequencing Method Improves Clinical Genomic Breakpoint Detection, Carrier Screening

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NEW YORK – Researchers from the King Abdullah University of Science and Technology (KAUST) in Saudi Arabia and their collaborators have developed a nanopore sequencing assay that promises to pinpoint complex breakpoints in genomic disorders at base resolution with rapid turnaround and low cost.

Dubbed nanopore-based rapid acquisition of neighboring genomic regions (NanoRanger), the method can help reach precise molecular diagnoses for patients with Mendelian disorders and enable effective carrier testing, according to its developers.

Mo Li, a bioscience professor at KAUST whose team developed the method, said NanoRanger is a targeted sequencing approach that combines amplification of genomic regions suspected of harboring mutations, long-read nanopore sequencing, and data analysis using in-house-developed pipelines.

To start, genomic DNA is extracted from patient samples using conventional techniques. Neither ultrahigh molecular weight DNA nor cross-linking are required, Li said.

Next, the DNA is partially digested by restriction enzymes into fragments of different sizes, typically ranging from a few to tens of kilobases. The fragments are then circularized using DNA ligase under conditions strongly favoring intramolecular ligation.

The DNA circles next undergo inverse PCR amplification using primers designed to amplify a genomic region that is suspected of harboring the mutation. After that, the amplicons are profiled using nanopore long-read sequencing, and the data is analyzed using a software called pyNanoRanger.

Li said NanoRanger can be designed as a multiplex assay where multiple loci are analyzed simultaneously, for example, all known genes implicated in a particular genetic disease.

In a paper in Med published in July, Li's team showed that NanoRanger "greatly improves the success rate of resolving base-resolution breakpoints of complex genomic disorders."

Initially, the KAUST researchers applied NanoRanger to DNA from a 23-year-old male patient with Bardet-Biedl syndrome whose genomic breakpoint was not clearly characterized by an array-based genotyping assay. Using 100 ng of genomic DNA from the patient and his parents, NanoRanger identified a complex rearrangement comprising a 4.6 kb inversion, a 189 bp deletion, and a 4.1 kb deletion. The breakpoints were further verified by Sanger sequencing, the study authors noted.

Encouraged by the result, the authors further tested NanoRanger on a dozen more patients who had a range of recessive genetic disorders but lacked precise molecular diagnoses from other tests, such as short-read-based panel sequencing, exome sequencing, array-based genotyping, optical genome mapping, and multiplex ligation-dependent probe amplification (MLPA). NanoRanger identified base-resolution breakpoints in all cases.

Furthermore, the authors compared NanoRanger with adaptive sampling, a feature released by Oxford Nanopore Technologies that allows software-controlled selective sequencing of regions of interest during a sequencing run.

Li said the results showed that NanoRanger is "much more efficient" for breakpoint analysis compared with adaptive sampling, which sometimes reaches insufficient read counts to resolve structural variants. Additionally, adaptive sampling used approximately 1 μg of input DNA compared with 100 ng per NanoRanger assay. The turnaround time for the adaptive sampling workflow was about 48 hours compared to an hour using NanoRanger.

According to Yingzi Zhang, a Ph.D. student in Li's lab and the first author of the Med paper, the total sample preparation time for NanoRanger takes a little bit over five hours, and the cost of the assay is estimated to be $8.80 per sample.

After the samples are loaded on the nanopore sequencer, the results can be analyzed in real time by pyNanoRanger, which was optimized to handle NanoRanger sequencing data using publicly available pipelines, Zhang added.

The study used the Oxford Nanopore MinIon device for sequencing, but Li said the method could also run on higher-throughput nanopore sequencers like the PromethIon.

Given the fast turnaround time and low cost, Li believes the NanoRanger workflow can be harnessed as an effective carrier screening tool for Mendelian genetic disorders that are prevalent in the Middle East, such as sickle cell disease and HBB thalassemia. "In our case studies, there are several [patients who have] the most frequent genetic disorders in Saudi Arabia," Li said. "We can design panels to target these disorders."

NanoRanger bears some similarities with the AmplideX Nanopore Carrier Plus assay, developed by molecular diagnostics company Asuragen in collaboration with Oxford Nanopore and researchers from Al Jalila Children’s. That assay deploys targeted nanopore sequencing to profile 11 genes associated with nine common carrier screening conditions.

One current limitation of NanoRanger is that it is PCR-based, eliminating the epigenetic signatures of the genomic DNA samples. A future iteration of NanoRanger could enrich and sequence native DNA, Li said, which will preserve methylation information.

Moving forward, his team will continue to streamline the NanoRanger workflow, including incorporating automation for sample handling and data analysis, to help drive the method's clinical adoption. "We definitely want to work on further R&D to make this technology commercially accessible," he noted, adding that they are considering licensing the NanoRanger technology to others. 

Last month, Oxford Nanopore announced a memorandum of understanding (MoU) with KAUST "to collaborate on the research and development of advanced multiomics technologies."

While the announcement mentioned NanoRanger, a company spokesperson said Oxford Nanopore has no plans to commercialize the method.