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

Abstract 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 ( R 2 : 0.933–0.973, MAE: 3.18–5.18, RMSE: 4.71–7.27).