EMBL Australia Partner Laboratory Network, John Curtin School of Medical Research, The Australian National University, Canberra, Australia; The Shine-Dalgarno Centre for RNA Innovation, John Curtin School of Medical Research, The Australian National University, Canberra, Australia; The Centre for Computational Biomedical Sciences, John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
Department of Immunity, Inflammation and Infection, The John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia; Centre for Personalised Immunology, NHMRC Centre for Research Excellence, Australian National University, Canberra, ACT 2601, Australia.
Forensic Sci Int Genet. 2024 Jul;71:103048. doi: 10.1016/j.fsigen.2024.103048. Epub 2024 Apr 15.
DNA methylation plays essential roles in regulating physiological processes, from tissue and organ development to gene expression and aging processes and has emerged as a widely used biomarker for the identification of body fluids and age prediction. Currently, methylation markers are targeted independently at specific CpG sites as part of a multiplexed assay rather than through a unified assay. Methylation detection is also dependent on divergent methodologies, ranging from enzyme digestion and affinity enrichment to bisulfite treatment, alongside various technologies for high-throughput profiling, including microarray and sequencing. In this pilot study, we test the simultaneous identification of age-associated and body fluid-specific methylation markers using a single technology, nanopore adaptive sampling. This innovative approach enables the profiling of multiple CpG marker sites across entire gene regions from a single sample without the need for specialized DNA preparation or additional biochemical treatments. Our study demonstrates that adaptive sampling achieves sufficient coverage in regions of interest to accurately determine the methylation status, shows a robust consistency with whole-genome bisulfite sequencing data, and corroborates known CpG markers of age and body fluids. Our work also resulted in the identification of new sites strongly correlated with age, suggesting new possible age methylation markers. This study lays the groundwork for the systematic development of nanopore-based methodologies in both age prediction and body fluid identification, highlighting the feasibility and potential of nanopore adaptive sampling while acknowledging the need for further validation and expansion in future research.
DNA 甲基化在调节生理过程中起着至关重要的作用，从组织和器官发育到基因表达和衰老过程，并且已经成为鉴定体液和预测年龄的广泛使用的生物标志物。目前，甲基化标志物作为多重分析的一部分独立靶向特定的 CpG 位点，而不是通过统一的分析。甲基化检测也依赖于不同的方法学，从酶消化和亲和富集到亚硫酸氢盐处理，以及各种高通量分析技术，包括微阵列和测序。在这项初步研究中，我们使用单一技术——纳米孔自适应采样来测试同时识别与年龄相关和体液特异性的甲基化标志物。这种创新方法能够在单个样本中对整个基因区域的多个 CpG 标记位点进行分析，而无需专门的 DNA 制备或额外的生化处理。我们的研究表明，自适应采样能够在感兴趣的区域提供足够的覆盖范围，以准确确定甲基化状态，与全基因组亚硫酸氢盐测序数据具有很强的一致性，并证实了年龄和体液的已知 CpG 标记物。我们的工作还确定了与年龄强烈相关的新位点，提示了新的可能的年龄甲基化标记物。这项研究为基于纳米孔的方法在年龄预测和体液识别中的系统开发奠定了基础，突出了纳米孔自适应采样的可行性和潜力，同时承认在未来的研究中需要进一步验证和扩展。
