Body fluids are critical biological evidence for crime-scene reconstruction. Skins play a pivotal role in distinguishing intimate from social interactions in contested sexual assault cases as a common biosample. DNA methylation has emerged as a promising biomarker owing to its high specificity and stability in body fluid identification. Forensic scientists have identified body fluid-specific CpG markers for common body fluids (blood, semen, saliva, vaginal secretions, and menstrual blood) in forensic practice and established highly sensitive and stable multiplex systems. However, the application of a DNA methylation multiplex system for skin identification has not yet been reported. In this study, we systematically screened body fluid-specific CpG sites for skin, peripheral blood, saliva, semen, and vaginal secretions based on the Illumina Infinium Methylation EPIC BeadChip (850K) array data. A series of |Δβ| thresholds were evaluated for CpG sites selection, and 201 skin-specific CpG sites were screened out when threshold of |Δβ| = 0.5. Subsequently, 10 CpG sites were selected with favorable specificity and amplification efficiency after agarose gel electrophoresis and sanger sequencing validation to construct the multiplex system for skin identification. The developed multiplex system was evaluated in 160 body fluid samples, confirming its favorable specificity. It achieved a sensitivity of 0.25 ng and was further validated using simulated DNA mixtures, degraded skin DNA samples, and a shaving debris sample. In addition, a random forest model was constructed for skin identification. The results showed that skin components in simulated mixtures and degraded samples were correctly identified with 100% accuracy by combining the established multiplex system with the random forest model. These findings support the utility of this multiplex system as an efficient and reliable tool for forensic skin identification.

Authors

• Qiuyun Yang (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Hebin Wang (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Shanshan Wei (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Shengqiu Qu (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Weibo Liang (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)