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A data‐driven, high‐throughput methodology to determine tissue‐specific differentially methylated regions able to discriminate body fluids
Electrophoresis ( IF 3.0 ) Pub Date : 2021-02-11 , DOI: 10.1002/elps.202000217
Joana Antunes 1 , Quentin Gauthier 1 , Vanessa Aguiar-Pulido 2 , George Duncan 1 , Bruce McCord 1
Affiliation  

Tissue‐specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high‐resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.

中文翻译:

一种数据驱动的高通量方法,用于确定能够区分体液的组织特异性差异甲基化区域

组织特异性差异甲基化区域 (tDMR) 是基因组中具有调节这些组织类型中基因表达的甲基化模式的区域。在法医证据中检测 tDMR 可以识别痕量的体液。在本报告中,我们对现有阵列数据集进行了生物信息学分析,以确定是否可以从法医证据中识别出新的 tDMR 用于体液识别。一旦确定了这些位点,就设计了引物并进行了亚硫酸氢盐修饰。然后使用具有高分辨率熔解分析 (HRM) 的 qPCR 确定给定基因座处每种体液的相对甲基化水平。在对多种体液中的 127 种 tDMR 进行筛查后,我们能够识别出四种能够区分血液的新标志物(2 个标志物),阴道上皮细胞(1 个标记)和颊细胞(1 个标记)。还用焦磷酸测序测试了每种目标体液的一个标记,显示的结果与 HRM 获得的结果一致。这项工作成功地证明了计算机分析,以开发一组能够通过实时 PCR/HRM 进行区分的新型 tDMR。该方法可以快速确定犯罪现场留下的体液,协助法医案件的事实审判者。
更新日期:2021-02-11
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