A comparative study of DNA conversion techniques: Assessing efficiency and sample suitability of enzymatic and...

Bisulfite conversion, the conventional method for DNA methylation analysis, converts unmethylated cytosine to uracil while leaving methylated cytosine unchanged. A recently developed enzymatic conversion method employing mild reaction conditions produces DNA sequences identical to those generated by bisulfite treatment. In this method, methylated cytosine is oxidized to 5-carboxycytosine (5caC) by ten-eleven translocation dioxygenase 2 (TET2). Subsequently, cytosine is deaminated by the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC), without affecting 5caC. These gentle reaction conditions show promise for identifying challenging forensic samples. Herein, we applied the enzymatic conversion method for the identification of body fluids. We developed a multiplex DNA methylation-based detection system and conducted a systematic comparison between enzymatic and bisulfite conversion methods. Parameters assessed included conversion efficiency, recovery, methylation detection at target CpG sites, and performance with degraded and mock casework samples. In addition, classification models were employed to enhance the interpretation of methylation data. Our findings demonstrated that the enzymatic conversion method exhibited lower conversion efficiency and recovery rate than bisulfite treatment. However, only marginal differences were observed in the accuracy of body fluid identification between the two methods. Enzymatic conversion preserved a greater number of CpG sites than bisulfite treatment in degraded samples. Moreover, when the DNA input was 10 ng, a decline in result stability was observed in bisulfite-treated samples. These results suggest that the enzymatic conversion method holds potential for challenging forensic sample identification.