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P153 – A Multi-CpG-microhaplotype Composite Marker System for Simultaneous Body Fluid Identification and Contributor Association in Body Fluid Mixtures
Body fluid mixtures are common challenging biological samples, particularly those containing semen and vaginal fluid. In forensic practice, the ability to simultaneously identify the body fluid type and infer its contributor in mixtures is critical for evidence interpretation. However, achieving this goal remains a major challenge. In this study, we proposed a novel composite marker, the multi-CpG-microhaplotype, which integrates a multi-CpG region composed of adjacent CpG sites with a nearby microhaplotype within the same short DNA fragment. Compared with single CpG sites, the multi-CpG region provides more stable and specific body fluid information, whereas microhaplotypes exhibit high polymorphism and are well suited for individual identification. After screening, 14 multi-CpG-microhaplotype loci were selected, including 9 semen-specific loci and 5 vaginal fluid-specific loci. Based on these loci, a multiplex bisulfite sequencing system was established to simultaneously detect multi-CpG methylation patterns (fully continuous methylation/unmethylation ratios) and microhaplotype genotypes (alleles) from the same amplicons. The developed system achieved a combined power of discrimination of 0.999999999927733. For mixture interpretation, a random forest-based allele classification model was developed using allele-level multi-CpG methylation patterns to identify alleles derived from the target body fluid. After allele classification, an improved random man not excluded method was applied to associate the target body fluid with its potential contributor. All tested mixtures were successfully identified as containing the target body fluid. The semen-specific loci correctly associated semen with its true contributor in semen:vaginal fluid mixtures at ratios of 19:1, 4:1, 1:1, 1:4, and 1:19. The vaginal fluid-specific loci correctly associated vaginal fluid with its true contributor in semen:vaginal fluid mixtures at ratios of 19:1, 4:1, 1:1, 1:4, and 1:19. These results demonstrate that multi-CpG-microhaplotypes provide an effective framework for simultaneous body fluid identification and contributor association in forensic mixtures.
Authors
- Dan Wen (Central South University, China)
- Xuan Tang (Central South University, China)
- Yue Wang (Central South University, China)
- Yajie Ma (Central South University, China)
- Zhuanxia Liu (Central South University, China)
- Weifeng Qu (Central South University, China)
- Ying Liu (Central South University, China)
- Lagabaiyila Zha (Central South University, China)
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