HGSA 2026

Background: Current hereditary colorectal cancer multigene panel testing uses conventional short-read next-generation sequencing, which can miss complex genetic variations such as structural variants (SVs) and single nucleotide variants (SNVs) in repetitive regions. We aimed to assess diagnostically challenging hereditary colorectal cancer and polyposis patients with unexplained aetiology using Oxford Nanopore Technologies (ONT) long-read sequencing. Methods: We performed ONT Adaptive Sampling of 385 known hereditary cancer genes in 16 colorectal cancer and polyposis patients without known germline pathogenic variants (PVs). This included unexplained adenomatous polyposis (UAP, n = 11), unexplained hamartomatous polyposis (n = 2 family members), familial colorectal cancer Type X (FCCТX, n = 2 family members), suspected Lynch syndrome (n = 4), and serrated polyposis syndrome (SPS, n = 4). All UAP cases had previously tested negative for APC mosaicism. Basecalling was performed using the Dorado super accuracy model and variants were called using the wf-human-variation workflow incorporating Sniffles2 and Clair3. Results: ONT sequencing identified a novel predicted loss-of-function APC intronic variant (NM_000038.6: c.1958+428G>T) in one proband and the relative with UAP. The SpliceAI in silico tool predicted this variant to create an abnormal acceptor splicing site (score = 0.25). The variant was absent from the gnomAD database and overlapped a 300 bp short interspersed nuclear element. No SVs or predicted PVs were identified in the remaining UAP cases. One SPS patient was identified with a novel 6.5 kb deletion in RNF43. One suspected Lynch syndrome family was identified with a 3.2 kb SVA insertion in MSH2. No SVs or complex predicted PVs in known CRC and polyposis genes were identified in the unexplained hamartomatous polyposis or FCCTX cases. Conclusions: ONT long-read sequencing technology provides an accurate, cost- and time-effective approach to detect complex genetic variants in diagnostically challenging hereditary colorectal cancer and polyposis syndrome patients.

Accurate haplotype phasing is critical for clinical variant interpretation, particularly for resolving cis/trans relationships in recessive Mendelian disease. Long-read sequencing from Oxford Nanopore Technologies (ONT) enables direct haplotype reconstruction, yet its performance across clinically relevant genomic regions has not been comprehensively evaluated. We evaluated ONT phasing performance in the HG002 (NA24385) reference sample by benchmarking against Genome in a Bottle (GIAB) high-confidence phased variant calls (v4.2.1). Accuracy was assessed using pairwise and per-variant metrics. To extend beyond GIAB high-confidence regions, trio-based phasing was performed in 10 clinical samples and one HG002 GIAB sample to generate independent truth sets, and accuracy was evaluated in phased replicates using the same framework. Phasing completeness was quantified across autosomes and within clinically relevant genes in 21 GIAB replicates and 34 clinical samples. Associations of phasing completeness with sequencing and variant characteristics, including read length, coverage, fragment length, and heterozygosity, were examined. ClinVar pathogenic and likely pathogenic variants were analysed to evaluate clinical interpretability. ONT achieved high phasing accuracy, with per-variant and pairwise accuracies of approximately 98% and 97% in HG002, and 97% and 94% in clinical samples, respectively. Median phased autosomal coverage reached 84% in GIAB samples and 88% in clinical samples, with comparable completeness across clinically relevant genes. Phasing completeness was strongly associated with read length N50 and heterozygosity. Pathogenic and likely pathogenic ClinVar variants showed consistently high phasing rates, with a median of at least 80% variants phased across both clinical and GIAB samples. Although not sufficient to fully replace trio-sequencing, even for recessive conditions, ONT long-read sequencing provides accurate and contiguous haplotype phasing across a majority of clinically relevant regions. Importantly, substantial phasing can be achieved using standard DNA extraction and routine library preparation protocols, supporting feasibility for integration into routine clinical genomic workflows requiring robust haplotype resolution.