Abstract: The identification of tissue origin of body fluid can provide clues and evidence for criminal case investigations. To establish an efficient method for identifying body fluid in forensic cases, eight novel body fluid-specific DNA methylation markers were selected in this study, and a multiplex single base extension reaction (SNaPshot) system for these markers was constructed for the identification of five common body fluids (venous blood, saliva, menstrual blood, vaginal fluid, and semen). The results indicated that the in-house system showed good species specificity, sensitivity, and ability to identify mixed biological samples. At the same time, an artificial body fluid prediction model and two machine learning prediction models based on the support vector machine (SVM) and random forest (RF) algorithms were constructed using previous research data, and these models were validated using the detection data obtained in this study (n=95). The accuracy of the prediction model based on experience was 95.79%; the prediction accuracy of the SVM prediction model was 100.00% for four kinds of body fluids except saliva (96.84%); and the prediction accuracy of the RF prediction model was 100.00% for all five kinds of body fluids. In conclusion, the in-house SNaPshot system and RF prediction model could achieve accurate tissue origin identification of body fluids.

Key words: DNA methylation; Body fluid; Forensic identification; Single base extension reaction (SNaPshot); Machine learning

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