Nanopore-Based DNA Methylation: Advancing Forensic Age and Body Fluid Analysis
Organized by: International Forensic Scientist Awards
Website: forensicscientist.org
15th Edition of Forensic Scientist Awards 27-28 October 2025 | Paris, France
Forensic DNA phenotyping is revolutionizing crime scene investigations by using genetic and epigenetic markers to infer biological age, biogeographical ancestry, and physical traits—especially in cases where conventional identification methods fall short. Among these markers, DNA methylation stands out due to its tissue-specific patterns, which allow not only accurate age estimation but also precise body fluid identification. This capability is crucial for reconstructing crime scenes and linking evidence to individuals.
Traditional methods for DNA methylation analysis using massive parallel sequencing (MPS) involve complex, multi-step protocols that are often time-consuming. In contrast, Nanopore sequencing offers a rapid and efficient alternative, enabling direct, real-time detection of DNA methylation without the need for DNA conversion. Our study explored the capabilities of Nanopore’s PromethION 2 platform for a comprehensive single-assay forensic approach, analyzing both epigenetic clocks for age estimation and markers for body fluid identification, even from low DNA quantities (<100 ng).
We observed that low read depth coverage sometimes produced extreme beta values (0 or 1), reflecting challenges in accurately measuring methylation’s continuous nature. This affected age prediction models, which initially tended to overestimate ages. By applying a proof-of-concept linear correction model, we significantly improved prediction accuracy. Body fluid identification, particularly for blood and saliva, remained highly accurate across both high and low read depth conditions, successfully identifying all samples in each experiment.
This exploratory work highlights the potential of adaptive sampling on the PromethION platform for forensic applications. Future research will focus on validating analysis thresholds, enhancing performance with low-quantity samples, expanding body fluid identification to more tissue types, and improving mixture analysis techniques. Nanopore-based methylation analysis holds great promise for making forensic investigations faster, more precise, and more informative than ever before.
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