Exploratory study on source identification of saliva stain and its TsD inference based on the microbial relative and absolute abundance.

In recent years, it has become a major research trend to obtain the microbial relative abundance in common body fluid stains at the crime scenes through 16S rRNA next generation sequencing to explore the effectiveness in forensic application. However, few scholars have combined the determination of tissue sources of body fluid stains with the inference of time since deposition (TsD) based on the relative and absolute abundance of microorganism in the same sample in a single study. Therefore, we preliminarily used the four abundant saliva-related bacteria to distinguish fresh saliva, saliva stains (exposure ≤60 days) from the four kinds of fresh body fluids and epidermal tissue, simultaneously assessed the temporal variation regularities in both microbial relative and absolute abundance in these saliva stains. Quantitative real-time PCR results demonstrated that fresh saliva samples and saliva stains exposed for up to 60 days still retained two or more abundant saliva-related bacteria, demonstrating sufficient discriminative power to identify saliva stain from other four kinds of body fluids and tissue. Microbial compositions and temporal analyses of 56 saliva samples revealed that many phyla and genera with abundance higher than 1% had different temporal variation regularities in relative and absolute abundance data, except for some genera such as Neisseria, etc. Beta diversity analysis indicated greater differences in absolute quantitative data among fresh saliva samples and saliva stains at different time points compared with relative quantitative data. The support vector machine (svm) model based on microbial relative or absolute abundance both have the prediction accuracy higher than 0.8 in classifying saliva stains deposited at 1 h, 1 day, and 7 to 60 days. This study combined the tissue origin identification and TsD inference of saliva stains, and the absolute quantitative technology was applied for the first time to the TsD inference of saliva stains. And the results indicated that using the absolute quantitative technology might be more suitable for early TsD inference (within 14 days) of saliva stains in this study, which helped to accurately infer the TsD of saliva stains, providing an important clue for forensic investigation.