P239 – A Highly Integrated Next-generation Forensic DNA and DNA Methylation Sequencing Experimental Workflow

11:01

Thursday August 20^th

Station 16

Duration: 12 minutes

04. Forensic biology

Le Wang

DNA genetic markers (STRs, SNPs) are essential for forensic identification, while DNA methylation markers excel in age estimation and tissue typing. Traditionally, analyzing these markers requires separate workflows and dual DNA aliquots. The standard bisulfite conversion method for methylation alters ~96% of cytosines, significantly disrupting genomic sequences and GC content, thereby hindering integrated analysis. Additionally, its harsh conditions can damage DNA samples. To address these limitations, we developed a highly integrated next-generation sequencing workflow using an enzyme-based methylation conversion method. Unlike bisulfite treatment, this enzymatic approach converts only the ~4% of methylated cytosines to thymines, minimizing impact on the overall DNA sequence. We designed a specialized assay panel enabling single-tube multiplex analysis of 55 SNP genetic markers and 11 CpG methylation markers. The SNP panel achieves a cumulative discrimination power of 1−2.18×10−21, ensuring robust individual identification. Six CpG markers accurately distinguish three common body fluids: peripheral blood, semen, and vaginal secretions. Validation using 21 samples (seven of each fluid type) yielded results consistent with expectations. An age prediction model for peripheral blood samples, built on five age-specific methylation sites from 30 individuals (aged 18–74), demonstrated a mean absolute error of 4.53 years upon cross-validation. Sensitivity tests confirmed the system’s efficacy with as little as 2 ng of input genomic DNA or 0.1 ng of converted DNA, maintaining precise genotyping and fluid identification. Cross-platform validation between MiSeq and GeneMind sequencers showed 100% concordance for SNP genotyping and a high correlation ( r=0.9772 ) for CpG methylation levels, confirming excellent reproducibility. This integrated approach significantly reduces sample consumption and simplifies experimental procedures compared to traditional methods. It provides a powerful, unified tool for comprehensive forensic genetic and epigenetic analysis, particularly valuable for challenging casework samples.

Authors

Le Wang (Institute of Forensic Science, Ministry of Public Security, China, China)
Na Yi (Institute of Forensic Science, Ministry of Public Security, China, China)
Hui Xu (Institute of Forensic Science, Ministry of Public Security, China, Afghanistan)
Guang-Bin Zhao (Institute of Forensic Science, Ministry of Public Security, China, China)
Chi Zhang (Institute of Forensic Science, Ministry of Public Security, China, China)
Ke-Lai Kang (Institute of Forensic Science, Ministry of Public Security, China, China)

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P239 – A Highly Integrated Next-generation Forensic DNA and DNA Methylation Sequencing Experimental Workflow

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