Comparison of Four Bisulfite Conversion Kits and Their Effects on DNA Methylation Detection and Age Prediction

Background/Objectives: This study aimed to compare the performance of four commercial bisulfite conversion kits-the Premium Bisulfite Kit, the SuperMethyl™ Fast Bisulfite Conversion Kit, the SuperMethyl™ Max Bisulfite Conversion Kit, and the EZ DNA Methylation-Gold™ Kit (hereafter: Premium, Fast, Max, and Gold)-to evaluate their impact on the age prediction accuracy at different DNA input levels. Methods: Comparisons were performed using DNA extracted from blood samples and DNA methylation standards. Different inputs of DNA were tested (1-50 ng), and all samples were analyzed twice. After bisulfite treatment, 10 targets were amplified by PCR and sequenced on a MiSeq instrument. Performance was evaluated across several metrics: DNA yield after PCR, conversion efficiency, read depth and β-value reproducibility, β-value specificity, cost, and hands-on time. Furthermore, DNA methylation levels of age-associated CpGs on the 10 PCR fragments were investigated, and the accuracy of age predictions was determined. Results: No kit was superior in performance across all metrics. Higher post-PCR DNA concentrations and more robust and reproducible results, particularly at lower DNA inputs, were obtained after treatment with the Premium and Max kits. The amplicon concentrations, read depths, and reproducibility between duplicate determinations were significantly lower with 1 ng DNA input compared to inputs of 5-50 ng DNA. Conclusions: For forensic genetic age prediction purposes, the Max or Premium kits represented more suitable options for bisulfite conversion, whereas the Fast kit may be preferable in settings where sample quantity is not a limiting factor, and cost or throughput are the primary considerations.