Age-associated DNA methylation loci at lncRNA genomic regions revealed by Oxford Nanopore whole-genome sequencing using four methylation callers

Accurate age inference from biological material is an essential component of forensic investigations. DNA methylation is a particularly informative biomarker, yet most current forensic approaches rely on bisulphite conversion, which can degrade DNA and limit applicability to compromised samples. Oxford Nanopore Technologies (ONT) enables direct detection of methylated cytosines from native DNA and therefore represents a promising alternative for epigenetic age inference. In this study, whole-genome ONT sequencing was performed on buccal DNA samples from 26 individuals aged 21-77 years to identify novel age-associated methylation signatures beyond previously established CpG markers. Sequencing was conducted on MinION R9.4.1 flow cells with Remora-optimised GPU basecalling, followed by alignment to GRCh38 and read-level quality filtering (Q ≥ 10). DNA methylation was profiled using four independent tools (Nanopolish, DeepSignal, Megalodon and Remora). To maximise reliability, we applied a stringent coverage filter, retaining only sites with at least 100 reads per site read depth, and considered as candidates only those loci that showed consistent age-methylation trends across all four methods. Under these conditions, multiple genomic coordinates exhibited strong correlations with chronological age (R² > 0.95). Strikingly, a substantial proportion of these high-confidence sites clustered within the same lncRNA loci on chromosome 21 (ENSG00000280441, ENSG00000278996), indicating that DNA methylation at lncRNA genomic loci changes is reproducibly detected even after rigorous quality and coverage filtering. Additional highly correlated sites were located in unannotated genomic regions, suggesting the presence of previously unrecognised epigenetic elements. These results expand the catalogue of candidate age-associated methylation markers, highlight the robustness of lncRNA-linked signals under strict bioinformatic filtering, and underscore the value of ONT sequencing for genome-wide epigenetic discovery in forensic age inference.