Forensic research surrounding the use of DNA methylation (DNAm) markers to predict age suggests that accurate prediction of chronological age can be achieved with just several DNAm markers. Several age-prediction models are based on DNAm levels that are detectable by a diverse range of DNAm analysis methods. Among the many DNAm analysis methods, targeted amplicon-based massively parallel sequencing (MPS) and single-base extension (SBE) methods have been widely studied owing to their practicality, including their multiplex capabilities. Since these two DNAm analysis methods share an identical amplification step during their experimental processes, several studies have compared the differences between the methods to construct integrated age-prediction models based on both MPS and SBE data. In this study, we compared the specific differences in DNAm levels between these two commonly exploited analysis methods by analyzing the identical PCR amplicons from the same samples and quantifying the actual bisulfite-converted DNA amount involved in the PCR step. The DNAm levels of five well-studied age-associated markers-CpGs on the ELOVL2, FHL2, KLF14, MIR29B2CHG, and TRIM59 genes-were obtained from blood samples of 250 Koreans using both DNAm analysis methods. The results showed that only ELOVL2 is interchangeable between the MPS and SBE methods, while the rest of the markers showed significant differences in DNAm values. These differences may result in high errors and consequential lowered accuracy in age estimates. Therefore, a DNAm analysis method-specific approach that considers method-induced DNAm differences is recommended to improve the overall accuracy and reliability of age-prediction methods.
Keywords: Age correlation; DNA methylation; Massively parallel sequencing; Multiplex PCR; Single-base extension.
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