Evaluation of DNA methylation markers for forensic applications

Abstract

DNA methylation analysis displays considerable promise in forensic applications offering additional information to traditional DNA profiling techniques. This research assesses the suitability and feasibility of DNA methylation based chronological age estimation and identical twin differentiation in the New Zealand population. It also seeks to address knowledge gaps in the field by identifying additional age-associated CpG markers for semen samples, as well as exploring the potential of a male age estimation assay for use in mixtures. Candidate CpG sites were identified from the literature and through the analysis of publicly available microarray datasets. Multiplex assays for age estimation in blood, semen and buccal samples, male-specific age estimation and identical twin differentiation were designed and optimised for targeted bisulphite massively parallel sequencing. Following the analysis of 170 blood and 250 buccal samples, age prediction models were developed achieving an average error of 2.1 years for blood samples and 3.3 years for buccal samples. Development of male age estimation models using 50 male blood and 90 male buccal samples achieved prediction accuracies of 9.7 years and 8.8 years respectively, providing some investigative value at a generational level. The accuracy of these models was retained down to 5 – 10 ng of starting DNA input and remained stable for samples stored for up to 24 weeks at room temperature. Importantly, the male estimation assays illustrated specificity when assessed with female samples. Analysis of nine semen samples highlighted the potential of accurate age estimation for this biological fluid once limitations regarding sample size have been overcome. These results demonstrate the suitability and feasibility of DNA methylation-based age estimation in forensic science. Additional efforts to improve the accuracy of the male age estimation models are necessary to increase the investigative value of this method. Six out of thirty sets of identical siblings displayed methylation level differences over 15% at one or more CpG sites in buccal samples. However, the number and magnitude of CpG site differences needed for robust individual identification remain unclear. These results demonstrate the feasibility of identical twin differentiation however the importance of appropriate characterisation of the variation present within samples from the same individual is highlighted.