An Exploratory Research For Identification Of Forensic Body Fluid Spots Based On DNA Methylation By Massively Parallel Sequencing

Background The tissue origin inference of body fluids based on tissue differential DNA methylation markers is a research hotpot in forensic medicine.The tissue specificity and stability of DNA methylation are important evaluation indicators for forensic application.The body fluid inference methods based on tissue differential methylation of single CpG sites showed high forensic application value.However,the large individual differences of methylation in some CpG sites reduced the accuracy of this method.The methylation haplotype in methylation regions showed higher tissue specificity and individual stability and could become better methylation markers for forensic body fluid identification.MPS is a technical method that can deeply analyze the differential methylation patterns in tissues,which can help to explore better body fluid identification markers.Objectives A methylation detection method based on MPS was established to explore the accuracy of different body fluid-specific methylation patterns.Methods A bisulfite-converted amplicon sequencing library was constructed for the sequencing of 41 body fluid-specific methylation fragments from 165 body fluid samples using Mi Seq double-end sequencing technology.The methylation analysis of the sequencing data was performed by using the bio-informatics analysis software,to obtain the methylation levels of the CpG sites of all fragments,and to screen the methylation markers with strong tissue specificity and good stability to establish a body fluid inference random forest mode.The best markers for body fluid identification were selected based on the accuracy of body fluid inference.Results The sequencing results of all the sample fragments were of good quality.The level of CpG site methylation in most of the fragments was highly consistent in each body fluid,showing significant differences between the target fluid and other body fluids.The accuracy of the body fluids inference reached 97.58% based on the methylation levels of ten sites in nine of these fragments and 98.18% based on the methylation levels of eighteen sites with stronger body fluid specificity in nine of these fragments.The inference of body fluid-specific discrimination based on the five specifically strongest CpG markers in all fragments reached 100% accuracy.Conclusions Methylation assay based on MPS screened out more fluid-specific methylation patterns.Haplotype markers composed of multiple tissue-specific methylation CpG sites could help to improve the accuracy of identifying body fluids based on DNA methylation.