An examination of differences in epigenetic methylation of saliva type samples based on collection method

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.