Analyzing variants and methylation in complex genomic regions using targeted long-read nanopore sequencing: A PKD case study.

Abstract

e22644 Background: In oncology, actionable genomic and epigenomic alterations often occur in repetitive or other structurally complex regions. However, many targeted methods rely on short reads or PCR amplification, limiting resolution. Methods that preserve long-read context and native epigenetic information are therefore needed to interrogate this complexity without sacrificing scalability or cost efficiency. Here, we describe an Agilent-enabled targeted nanopore sequencing workflow for simultaneous native variant and methylation analysis, using autosomal dominant polycystic kidney disease (ADPKD) as a case study for resolving methylation and challenging genomic architecture. Methods: Assay performance was evaluated using SureSelect-based probe sets targeting multiple genomic regions across Genome in a Bottle (GIAB) reference samples. Libraries were sequenced on Oxford Nanopore platforms and analyzed using an integrated workflow including alignment, variant calling, and methylation profiling. Clinical applicability was assessed using a targeted ADPKD panel evaluated in a clinically verified patient sample and HG002 GIAB controls. Results: Across 634 filtered target regions, single-nucleotide variant detection achieved a mean precision of 98.5% and recall of 99.5%, and average methylation calling precision of 91% relative to GIAB truth sets, with consistent enrichment and coverage. With 1µg gDNA input per sample and 8-plex, mean on-target coverage exceeded 120x across genes, with fold enrichment greater than 1,600x. Long reads accurately resolved PKD1 despite highly homologous pseudogenes. Target variant calls were completely concordant with known patient variants and GIAB references. Conclusions: Together, these results indicate that Agilent-enabled targeted nanopore sequencing resolves variants in complex genomic regions, including loci with high homology or limited PCR accessibility, while preserving native methylation. Ongoing work focuses on expanding target designs, incorporating additional genomic features, and evaluating performance in clinical oncology samples to develop panels that capture SNPs, STRs, and other oncology-related variants.