| Background In the process of case investigation,the residual source of tissue fluid at the scene of the crime plays an important role in helping criminal investigators rebuild the crime scene and infer the crime experience,which can provide important clues for the police to determine the nature of the crime.Traditional body fluid spot identification methods,such as immunology and biochemistry,have weak specificity and poor sensitivity.Although the detection method based on RNA molecule is better specific,it has high requirements for experimental testing and material conditions,which cannot be widely used in general forensic cases.The DNA methylation-based body fluid spot identification method has high sensitivity,high specificity advantages and is compatible with the working platform of the general forensic DNA laboratory,which is a good choice in the detection method of biological material.Relevant studies have shown that a large number of tissue-specific differentiated methylation regions(tissue-specific differentially methylated regions,t DMRs)exist in the human genome.By detecting the levels of methylation in these regions,we can identify different human tissues.However,due to certain methylation difference and crossover between body fluid group,methylation markers already reported in other groups may not be applicable to body fluid identification in local populations.Therefore,it is necessary to further analyze and verify the body fluid specificity of the currently reported DNA methylation markers in the Chinese population,establish a DNA methylation multiplex assay system suitable for the Chinese population,and evaluate the value of its forensic application.The preliminary study of this project verified the body fluid-specific methylation markers reported abroad in Chinese population,and 10 DNA methylation markers with strong body fluid specificity in Chinese population were preliminarily selected.This paper proposes to establish a composite detection system based on ten body fluid-specific methylation markers,further verify the body fluid specificity of these markers in a large number of body fluid samples,and study the forensic value of the multiplex assay for the tissue sources identification of body fluid spot based on the analysis method of random forest model.Objective This study aims to establish a new multiplex assay system based on ten methylation markers,and to study the forensic value of the assay method for body fluid identification in the Chinese population.Methods Based on ten fluid-specific methylation Cp G markers selected by the research group,a SNa Pshot multiplex assay system was established.265 body fluid spot samples(50 semen samples,55 blood samples,52 saliva samples,56 vaginal secretions and 52 menstrual blood samples)from the Chinese Han population were tested,the methylation values of ten Cp G sites were obtained from the body fluid samples in the local population.A random forest model was established on the base of the methylation data of the tested samples,and was verified the accuracy of inferring the source of body fluid spot.The sensitivity of the multiplex assay system and random forest analysis method to infer the source of single body fluid spot tissue was test,and its ability to detect and infer the tissue origin of mixed fluid samples and old samples was also investigated in this study.Results This study successfully established a SNa Pshot multiplex assay system of ten body fluid-specific methylation markers and obtained good typing results in all body fluid samples.The methylation levels of ten markers have strong body fluid specificity in all samples.The random forest model for the body fluid identification based on the methylation data of the test set could infer the tissue source of the testing samples with a judgment accuracy of up to 100%(185 / 185),infer the tissue source of the verification samples with a judgment accuracy of 97.5%(78 / 80),and infer the tissue source of all samples with a judgment accuracy of 99.25%(263 / 265).In addition,the model successfully predicted semen,blood,saliva,vaginal fluid,and menstrual blood with the average probability of up to 0.99,0.99,1.0,0.98,and 0.83 respectively.The detection and analysis method accurately obtained a good methylation profiles from the samples of low to 0.5 ng DNA,the six-month old samples and made an accurate judgment on the source of the sample tissue.Moreover,the detection analysis method can also obtain a clear methylation pattern from a mixed sample with a mixture as low to 1: 20,and make accurate judgment on the tissue source of the mixed sample as low to 1: 5.Conclusions A new body fluid-specific methylation multiplex assay and random forest model was established for body fluid identification in this study.The multiplex assay and the random forest model have high accuracy and sensitivity for tissue source inference of the single sample,old sample and mixed sample of forensic body fluid spot.Our study provides a new detection and analysis method for tissue source inference of forensic body fluid spot,which has good forensic value. |
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