Sequencing method matters: differential performance of DNA methylation data acquisition in epigenetic clock calibration

Age is an important trait, influencing an individual's physiology, behavior, and survival probability. Unfortunately, the ages of wild animals are often unknown, impeding behavioral and ecological studies requiring life history information and impacting conservation efforts for threatened species. Epigenetic clocks offer a solution by generating accurate age estimates based on DNA methylation at particular CpG sites, which changes in a predictable manner over lifespan. The CpG sites correlating with age vary across species, requiring species-specific epigenetic clock calibration using DNA methylation measured at CpG sites across the genome. Here, we compared the efficacy of two DNA methylation sequencing methods, whole genome enzymatic methyl sequencing (WGEM-seq) and epiRADseq, in calibrating epigenetic clocks for chestnut-crowned babbler Pomatostomus ruficeps nestlings (n = 56, aged 0–19 days). We found that epigenetic clocks using WGEM-seq data outperformed clocks using epiRADseq data (MAE = 1.6 versus MAE = 5.9; r = 0.95 versus r = 0.41 for 10-fold cross-validation). Notably, WGEM-seq achieved superior performance despite utilizing fewer loci in the input data than epiRADseq, indicating that the lower resolution of epiRADseq does not allow for accurate clock calibration. Using WGEM-seq data collected throughout a small fraction of the lifespan of chestnut-crowned babblers, we successfully calibrated a highly accurate epigenetic clock. These results open promising avenues for investigating the impact of early life environments and developmental stressors on aging. The insights gained from such studies can improve conservation and management strategies while deepening our understanding of avian life history strategies, ecology, and behavior.