Designing an EpiTYPER bisulfite sequencing assay for age estimation in Acinonyx jubatus based on human orthologues

Age is key factor in animal ecology as it can be used to assign animals to important age classes, ranging from immature young to reproductive adults and eventual old age and fragility. Different groups contribute to different aspects that need to be considered when modeling current and future population dynamics as part of continued conservation efforts. Due to the need of an accurate molecular method for assigning age, several studies have explore various aspects of epigenetic clocks. Epigenetics is a collective term for mechanisms that modify DNA and DNA packaging, independent of genetic sequence. One widely studied epigenetic feature is DNA methylation; a process that adds a methyl group to the 5’ cytosine of Cytosine-Guanine pairs (CpG’s). Studies have revealed that within genes, nearly a third of all CpG sites are influenced by age. Given its consistency, the epigenetic clock is a promising avenue of chronological age prediction which has been illustrated in many human studies. This protocol illustrates how CpG's with known age-correlations from human studies can be used to (1) identify orthologous regions in other species and (2) design primers to assay differential methylation using EpiTYPER mass array technology.