Optimal sample size for calibrating DNA methylation age estimators

Age is a fundamental parameter in wildlife management as it is used to determine the risk of extinction, manage invasive species, and regulate sustainable harvest. In a broad variety of vertebrates species, age can be determined by measuring DNA methylation. Animals with known ages are initially required during development, calibration, and validation of these epigenetic clocks. However, wild animals with known ages are frequently difficult to obtain. Here, we perform Monte‐Carlo simulations to determine the optimal sample size required to create an accurate calibration model for age estimation by elastic net regression modelling of cytosine‐phosphate‐guanine methylation data. Our results suggest a minimum calibration population size of 70, but ideally 134 individuals or more for accurate and precise models. We also provide estimates to the extent a model can be extrapolated beyond a distribution of ages that was used during calibration. The findings can assist researchers to better design age estimation models and decide if their model is adequate for determining key population attributes.