Epigenetic differentiation of monozygotic twins in forensic investigations using nanopore sequencing

Monozygotic twins (MZTs) pose a significant challenge in forensic genetics due to their identical nuclear DNA, rendering conventional markers like STRs and SNPs ineffective. This limitation highlights the need for novel approaches to distinguish MZTs in criminal investigations, disaster victim identification, and paternity testing. Current methods lack the resolution to differentiate MZTs, creating a gap in human identification. Epigenetic markers, such as DNA methylation, offer a solution by reflecting environmental and stochastic differences post-twinning. However, traditional sequencing technologies often fail to meet forensic requirements for resolution, speed, or reliability. To address this, we employed Oxford Nanopore sequencing to analyze genome-wide DNA methylation patterns in six MZT pairs, identifying robust epigenetic biomarkers for forensic discrimination. Our approach leverages long-read sequencing and single-base resolution to overcome conventional limitations. The study aimed to identify differentially methylated loci (DMLs) as stable, heritable biomarkers for distinguishing MZTs, even in degraded or trace samples, while demonstrating the forensic utility of nanopore sequencing. We identified 3820 shared DMLs enriched in metabolic and neural pathways, localized to promoter (1.84 %) and intergenic (88.03 %) regions. Nanopore sequencing achieved > 99.5 % alignment efficiency and > 13 kb N50 read lengths, enabling rapid, PCR-free analysis. This study demonstrates that nanopore-based methylation profiling effectively distinguishes MZTs by capturing environmentally influenced epigenetic differences, providing actionable biomarkers for forensic discrimination. By bridging epigenetics and forensics, our findings advance precision in human identification, offering transformative tools for criminal casework, disaster victim identification, and paternity testing, addressing a longstanding limitation in resolving cases involving genetically indistinguishable individuals.