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. Epigenetic clocks offer a mechanism to estimate ages by generating accurate age estimates based on DNA methylation at particular CpG sites, which changes in a predictable manner over lifespan. 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). Epigenetic clocks using WGEM-seq data outperformed clocks using epiRADseq data, 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.