A cost-effective blood DNA methylation-based age estimation method in domestic cats, Tsushima leopard cats (Prionailurus bengalensis euptilurus), and Panthera species, using targeted bisulfite sequencing and machine learning models

Knowledge of individual age can help both in-situ and ex-situ conservation programs to design more efficient and suitable management plans for targeted wildlife species. DNA methylation is one of the epigenetic aging markers that has emerged as a promising tool that can estimate age with high accuracy using only a tiny amount of biological material, which can be collected in a minimally invasive way. Here, we sequenced five targeted genetic regions and used 8–23 selected CpG sites to build age estimation models with machine learning methods with about only $3–7 per sample, using blood samples of seven Felidae species—ranging from small to big, and domestic to endangered species: domestic cats (Felis catus, 139 samples), Tsushima leopard cats (Prionailurus bengalensis euptilurus, 84 samples), and five Panthera species (96 samples). The models built achieved satisfactory accuracy—the mean absolute error of the best models was 1.966, 1.348, and 1.552 years in domestic cats, Tsushima leopar..., , , # Datasets

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### Appendix S1–S4

The files included the methylation data, sample information, and predicted age of each target species/species group. The data in the files are used to build age estimation models. 'domestic cat' in the filename means the file is for the domestic cat; 'leopard cat' means for the Tsushima leopard cat; 'panthera' means for the Panthera species (i.e., jaguar, leopard, lion, snow leopard, and tiger), and 'all' means for all the samples from all species.

### Appendix S5

The file contains the CpG selection results for the best age estimation model of each species/species group, the frequency of being selected in elastic net feature selection of each CpG site, correlation coefficients between the methylation rate and chronological age of each CpG site, and NCBI sequence ID with position.

### CpG No renamed fulllist\_all felidae.csv

The file showed the list of CpGs, which were at least contained in one species.

### M%+sampleinfo\*.csv

These files are th...