Oxford Nanopore User Group Meeting, Hong Kong

X-linked genetic diseases affect sexes differently due to X-chromosome inactivation-driven differential methylation in females. Targeted long-read sequencing (LRS) via adaptive sampling (AS) characterizes genomic composition and methylation, but performs poorly with non-high-molecular-weight (non-HMW) DNA. We optimized an AS-based LRS protocol for non-HMW DNA and validated its diagnostic utility in 20 families with known X-linked defects previously identified by routine methods. Incorporating optimized DNA fragmentation, size selection, and library construction, our protocol achieved a >15-fold target enrichment compared to 8.1-fold using standard methods. The optimized LRS successfully detected all previously reported variants and uncovered novel genomic findings in 7/20 families (35%). By resolving structural variant breakpoints and determining gene methylation status, this protocol led to variant reclassification in 3/20 cases (15%). Ultimately, this optimized AS-based approach enables comprehensive genomic and methylation profiling of non-HMW DNA, offering a robust tool for evaluating X-linked defects with potential application to other disease states.

In clinical practice, we often encounter challenging cases in which contemporary techniques cannot always provide a complete diagnosis. This challenge is especially relevant to inherited disorders, for which the exact genetic aetiology may remain unknown even after a lengthy diagnostic odyssey. Recent advances in sequencing technologies have created new approaches to investigating genomic variation.

Driven by unmet diagnostic needs, we began developing and evaluating nanopore long-read sequencing capability at Princess Margaret Hospital. This session focuses on our experience with Flongle, Oxford Nanopore Technologies' compact, lower-cost flow-cell format, suited to focused, small-batch applications. Our long-read workflows characterised complex structural variants and evaluated repeat sizing and sequence interruptions in a proof-of-concept study of trinucleotide-repeat disorders. Ongoing analytical work extends to near-whole mitochondrial DNA sequencing for heteroplasmy measurement and large-deletion feasibility, and consensus-based ITS sequencing of survey and reference mushrooms for potential toxicology support.

Flongle's small-scale format provides a practical entry point for developing focused long-read applications. These examples demonstrate how we developed laboratory methods, bioinformatics workflows and interpretive experience with long-read sequencing. As experience, evidence and demand grow, the capability built through these projects may support broader applications at greater scale.

Alport syndrome (AS) is a prevalent inherited kidney disorder mainly caused by mutations in COL4A3, COL4A4, and COL4A5 genes. To elucidate the genetic variants of AS, we implemented a sequential sequencing strategy within a Chinese cohort of 555 patients, comprising whole-exome sequencing (WES) for all participants, followed by whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and nanopore long-read sequencing (NLR-seq) for selected individuals. We identify 431 distinct variants in 509 (91.7%) patients, with 42.2% being novel. Beyond WES, additional sequencing approaches resolve 23 patients with noncoding, copy number, or structural variants. Notably, noncoding variants account for 16.2% of detected variants and exhibit ethnic-specific mutagenesis patterns. More interestingly, NLR-seq uncovers two novel types of structural variants, namely large insertions in intronic regions and complex duplication-inversion variants. This study provides deeper insights into the genetic architecture of AS and proposes a research paradigm for improving the genetic diagnosis of inherited diseases.

In this presentation, the translation of research-developed assays into laboratory-developed tests for pharmacogenomics (PGx) and CNS tumor classification through the clinical implementation of Oxford Nanopore sequencing in Thailand will be presented. The presentation will highlight how different implementation strategies are required for public and private hospitals, reflecting differences in clinical priorities, reimbursement models, laboratory infrastructure, and patient access. It will also discuss the multidisciplinary collaborations needed to bridge research, pathology, medical technologist, bioinformatics, and clinical care. Through real-world case studies, the audience will gain practical insights into assay development, validation, quality management, workflow integration, and service scale-up. This journey demonstrates how long-read sequencing can move beyond proof-of-concept to become a sustainable clinical service, delivering precision medicine that is both clinically impactful and accessible across diverse healthcare settings.