The 12 o'clock assay: an optimized dodecaplex droplet digital PCR assay for robust DNA methylation quantification and epigenetic clock-based age-predictions

DNA methylation quantification in age-prediction models relies on only a few techniques with inherent limitations and biases, reducing the accuracy and reproducibility. We developed a new droplet digital PCR assay format for accurate DNA methylation quantification, based on three LNA-containing hydrolysis probes to detect methylated and unmethylated alleles. Using a 12-plex assay we simultaneously quantified methylation at six CpGs (ASPA, C1orf132, CCDC102B, EDARADD, ELOVL2, FHL2) in 351 blood samples (age range: 0-95 years), showing strong correlations with age (|r|> 0.83). Several age-prediction models were developed, achieving high performance on testing sets (R²: 0.933-0.973, MAE: 3.18-5.18, RMSE: 4.71-7.27).