Trace evidence recovered from crime scenes is frequently a mixture of DNA from several individuals, and this is particularly common in sexual assault cases. Although probabilistic genotyping software allows mixture deconvolution, identifying the donor of a specific body fluid, such as saliva, is difficult. In sexual offence investigations, determining which body fluids are contributed by the donor is crucial. Consequently, a strategy that can assign donors to specific fluids is desirable. To specifically isolate the person of interest from such biological mixtures, we previously proposed a saliva-specific genotyping method that simultaneously analyzes saliva-specific (un)methylated CpG sites and nearby SNPs. However, methylation-based approaches require a relatively large amount of salivary DNA to yield reliable results, which is a disadvantage for some forensic samples.

We previously developed meta-CRISPR typing, in which Streptococcus CRISPR loci were amplified and sequenced on an Illumina MiSeq platform for personal identification. The diversity of Streptococcus CRISPRs, highly polymorphic repetitive elements found in bacterial DNA, is known to be highly individual-specific. In addition, Streptococcus is more abundant in saliva than in other body fluids such as semen, theoretically allowing the selective amplification of salivary Streptococcus CRISPR reads from mixed biological samples.

In this study, we introduce a new saliva-specific identification strategy based on metagenomic sequencing of CRISPR arrays. Quantification of Streptococcus CRISPRs and human DNA by qPCR revealed high CRISPRs/human ratio in saliva compared to semen, indicating feasibility of saliva-specific deconvolution. Metagenomic sequencing of Streptococcus CRISPR spacers was performed on the total DNA that had been extracted for personal identification. In the mixture experiments, the DNA extracted from a saliva-derived sample (containing both human and Streptococcus DNA) was diluted with semen-derived DNA. Even when the final mixture contained as little as 1:1000 of salivary human DNA (i.e., ≈0.001 ng of human DNA from the saliva within approximately 1 ng of human DNA from the semen), the metaCRISPR typing was still able to recover and type the Streptococcus spacers, demonstrating high analytical sensitivity. Although large validation studies are required, this proof-of-concept study suggests that metaCRISPR typing can improve the body fluid-specific deconvolution of complex biological mixtures.

Authors

• Kochi Toyomane (National Research Institute of Police Science, Japan)
• Tomofumi Shibata (National Research Institute of Police Science, Japan)
• Takashi Fukagawa (National Research Institute of Police Science, Japan)
• Ken Watanabe (National Research Institute of Police Science, Japan)
• Takayuki Yamagishi (National Research Institute of Police Science, Japan)
• Tomoko Akutsu (National Research Institute of Police Science, Japan)
• Junji Hosokawa-Muto (National Research Institute of Police Science, Japan)
• Kazumasa Sekiguchi (National Research Institute of Police Science, Japan)