Forensic genetics has advanced significantly with DNA technologies. Capillary electrophoresis (CE) enabled accurate short tandem repeat (STR) genotyping, which remains the gold standard in forensic casework. However, the integration of high‑throughput platforms has enabled more comprehensive analyses of complex forensic scenarios requiring additional genetic and epigenetic information. Next-generation sequencing (NGS) allows high-throughput analysis of diverse markers, including STRs, single nucleotide polymorphisms (SNPs), and microhaplotypes. DNA methylation can also be analyzed using NGS of C-to-T converted DNA, but the bisulfite conversion process may cause DNA degradation and introduce bias. Recently introduced third-generation sequencing technologies, including nanopore sequencing by Oxford Nanopore Technologies (ONT) and single-molecule real-time (SMRT) sequencing by Pacific Biosciences (PacBio), enable direct detection of 5-methylcytosine (5mC) without chemical conversion. SMRT sequencing generates highly accurate long reads called high-fidelity (HiFi) reads, improving alignment in repetitive regions and enabling structural variation analysis. In this study, we evaluated the applicability of SMRT sequencing for analysis of forensic genetic and epigenetic markers. A total of 11 samples derived from autopsy and biopsy (blood, brain, heart, muscle, and skin) were analyzed through HiFi sequencing, and data processing followed the PacBio reference pipeline. Sufficient coverage was obtained, and long-read STR analysis demonstrated the ability to distinguish iso-alleles, increasing discriminatory power. SNP analysis showed potential for inference of externally visible characteristics, ancestry, and distant kinship relevant to investigative genetic genealogy. In addition, most tissue-specific DNA methylation markers showed expected methylation patterns, but some exhibited atypical profiles in non-target tissues. For age-associated DNA methylation markers, both array-based epigenetic clocks and MPS-based VISAGE markers were applied, but predicted ages varied substantially between models. This pilot study demonstrates the potential of third-generation sequencing in forensic genetic and epigenetic analysis. However, high DNA input requirements, cost, and limited protocol flexibility remain challenges. Future studies should address these limitations and expand applications to complex forensic scenarios, including mixed and small amount of DNA samples.

Authors

• Soyi Park (Department of Forensic Medicine, Seoul National University College of Medicine, South Korea)
• Sang Un Park (Department of Forensic Medicine, Seoul National University College of Medicine, South Korea)
• Sangbo Lee (Department of Biomedical Systems Informatics, Yonsei University College of Medicine, South Korea)
• Dongwoo Park (Department of Biomedical Systems Informatics, Yonsei University College of Medicine, South Korea)
• Soo-Bin Yang (Department of Forensic Medicine, Seoul National University College of Medicine, South Korea)
• Taehyeon Kim (Department of Biomedical Systems Informatics, Yonsei University College of Medicine, South Korea)
• Sangwoo Kim (Department of Biomedical Systems Informatics, Yonsei University College of Medicine, South Korea)
• Hwan Young Lee (Department of Forensic Medicine, Seoul National University College of Medicine, South Korea)