In the context of forensic casework, it is imperative to both establish a DNA profile from biological specimens and accurately identify the specific bodily fluid source. To achieve this, DNA methylation markers have been developed for the differentiation of blood, semen, vaginal epithelial secretions, and saliva samples. Saliva, alternatively referred to as oral fluid, is recognized for its heterogeneous cellular composition, characterized by a mixture of epithelial, leukocytic, and bacterial cells. Consequently, our research has revealed variations in methylation percentages that correlate with the method employed for collecting saliva samples. To investigate these concepts, we scrutinized four CpG markers situated within or in proximity to the BCAS4, SLC12A8, SOX2OT, and FAM43A genes. Subsequently, we designed primers based on bioinformatically transformed reference sequences for these markers and rigorously assessed their quality by examining dimer and hairpin formation, melting temperature, and specificity. These loci were identified as saliva markers based on either buccal swabs or spit collection. Yet, there has been minimal or no research conducted to explore the variations in methylation between different collection methods. For this study, buccal, lip, tongue, spit, and nasal swabs were collected from 20 individuals (N = 100). Mock forensic samples, which include chewing gum (N = 10) and cigarettes (N = 10), were also tested. DNA was extracted, bisulfite converted, then amplified using in‐house designed assays, and pyrosequenced. The methylation levels were compared to other body fluids (semen, blood, vaginal epithelia, and menstrual blood [N = 32]). A total of 608 pyrosequencing results demonstrated that sampling location and collection method can greatly influence the level of methylation, highlighting the importance of examining multiple collection/deposition methods for body fluids when developing epigenetic markers.

中文翻译：

---

基于采集方法检测唾液型样本表观遗传甲基化差异

在法医案件工作中，必须从生物样本中建立 DNA 图谱并准确识别特定的体液来源。为了实现这一目标，我们开发了 DNA 甲基化标记来区分血液、精液、阴道上皮分泌物和唾液样本。唾液，也称为口腔液，因其异质细胞组成而闻名，其特征是上皮细胞、白细胞和细菌细胞的混合物。因此，我们的研究揭示了甲基化百分比的变化，这与收集唾液样本的方法相关。为了研究这些概念，我们仔细检查了位于 BCAS4、SLC12A8、SOX2OT 和 FAM43A 基因内部或附近的四个 CpG 标记。随后，我们根据这些标记物的生物信息转化参考序列设计了引物，并通过检查二聚体和发夹形成、解链温度和特异性来严格评估其质量。根据口腔拭子或唾液收集，这些位点被鉴定为唾液标记。然而，很少有或根本没有研究来探索不同收集方法之间甲基化的差异。在这项研究中，采集了 20 名个体的口腔、嘴唇、舌头、唾液和鼻拭子（氮= 100）。模拟法医样本，其中包括口香糖（氮= 10) 和香烟 (氮= 10），也进行了测试。提取 DNA，进行亚硫酸氢盐转化，然后使用内部设计的检测方法进行扩增，并进行焦磷酸测序。将甲基化水平与其他体液（精液、血液、阴道上皮和经血）进行比较[N=32]）。总共 608 项焦磷酸测序结果表明，采样位置和收集方法可以极大地影响甲基化水平，凸显了在开发表观遗传标记时检查体液多种收集/沉积方法的重要性。