Construction of the time since deposition (TsD) model in saliva stains with 16S rRNA full-length sequencing technology and microbial markers

Determining the time since deposition (TsD) and sex of saliva stains is crucial for revealing the time of the crime's occurrence and clarifying the nature of the crime. This process not only shortens the time required to solve the case but also helps narrow down the scope of investigation, thereby enhancing the efficiency of case resolution. Currently, the forensic study of the microbial composition in long-term saliva stains remains a relatively underexplored field. The purpose of this study was to explore the succession pattern of long-placed human saliva stains microbial communities and identify relevant microbial markers for estimating TsD and identifying the sex of the donor, in order to be an effective alternative tool for solving practical forensic cases. Therefore, in this study, saliva stains exposed to indoor environmental conditions for up to 140 days were collected and 16S rRNA full-length sequencing was performed using single-molecule real-time sequencing technology based on the PacBio sequencing platform. The study reveals that after 140 days of placement, the relative abundance of Firmicutes significantly decreased (p = 0.00304). At the genus level, the relative abundances of Streptococcus (p = 0.0008), Rothia (p = 0.0448), Gemella (p = 0.016), and Veillonella (p = 0.0208) also significantly decreased. Additionally, significant differences were found in the microbial communities between saliva stains from males and females (p = 0.00013). Then, we constructed a TsD estimating model for microbial community markers based on random forest, and the results showed that the mean absolute error was 9.59 days, and the accuracy of sex classification model based on stepwise logistic regression model and 4 bacterial markers was 84.21%. This indicates that saliva stains that have been in place for a long time still retain significant forensic value, and microbial markers can be used to determine the time since deposition (TsD) of dried saliva stains as well as to identify the sex of the donor.