In cases of sexual assault, the evidence often exists as a mixture of female and male body fluids, and in many cases, contains a higher proportion of female body fluids than males. In these cases, Y-STR, rather than autosomal STRs, can provide useful information. It becomes very difficult to identify the true suspect if there is no match among known suspects or if a match exists for two or more suspects, e.g. two suspects from the same paternal lineage. However, age prediction using the DNA methylation of Y-chromosomal CpGs can help narrow the search for unknown suspects and discriminate between older and younger suspects. Therefore, the DNA methylation profiles of semen samples from 56 healthy Korean males were generated using Illumina’s Infinium MethylationEPIC BeadChip Array. Among the ten identified age-associated CpG markers located in the Y-chromosome, nine were used to construct age prediction models. The identified markers were further investigated in the MPS analysis of 147 semen samples, and the multiplex assay was validated with the reliability, reproducibility and sensitivity tests. Several age prediction models were constructed using the MPS data with the multiple linear regression, stepwise linear regression, ridge linear regression, lasso regression, elastic net linear regression and support vector machine analyses, and all showed MAEs of 5 to 7 years in the test set samples. Six single-source female samples were also subjected to MPS analysis but showed very low coverage that could not affect the analysis of the mixed samples. Therefore, the age prediction models of the present study are expected to provide useful investigative leads, especially in mixed male and female samples from sexual assault cases.

在性侵犯案件中，证据通常以女性和男性体液的混合物形式存在，并且在许多情况下，女性体液的比例高于男性。在这些情况下，Y-STR（而不是常染色体 STR）可以提供有用的信息。如果已知嫌疑人之间不存在匹配，或者两个或多个嫌疑人存在匹配，例如来自同一父系的两个嫌疑人，则识别真正的嫌疑人变得非常困难。然而，利用 Y 染色体 CpG 的 DNA 甲基化进行年龄预测可以帮助缩小对未知嫌疑人的搜索范围，并区分年长和年轻的嫌疑人。因此，使用 Illumina 的Infinium MmethylationEPIC BeadChip Array生成了 56 名健康韩国男性精液样本的 DNA 甲基化谱。在位于 Y 染色体的 10 个已识别的年龄相关 CpG 标记中，有 9 个用于构建年龄预测模型。在 147 份精液样本的 MPS 分析中进一步研究了所识别的标记，并通过可靠性、再现性和敏感性测试验证了多重测定。使用MPS数据通过多元线性回归、逐步线性回归、岭线性回归、套索回归、弹性网络线性回归和支持向量机分析构建了多个年龄预测模型，并且在测试集中均显示出5至7年的MAE样品。六个单一来源的女性样本也接受了 MPS 分析，但覆盖率非常低，不会影响混合样本的分析。因此，本研究的年龄预测模型有望提供有用的调查线索，特别是在性侵犯案件的男女混合样本中。