DOI: 10.5061/dryad.qjq2bvqnb
title: Age estimation of captive Asian elephants (Elephas maximus) based on DNA methylation: An exploratory analysis using methylation-sensitive high-resolution melting (MS-HRM)

description: Age is an important parameter for bettering the understanding of
 biodemographic trends-development, survival, reproduction and
 environmental effects-critical for conservation. However, current age
 estimation methods are challenging to apply to many species, and no
 standardised technique has been adopted yet. This study examined the
 potential use of methylation-sensitive high-resolution melting (MS-HRM), a
 labour, time, and cost-effective method to estimate chronological age from
 DNA methylation in Asian elephants (Elephas maximus). The objective of
 this study was to investigate the accuracy and validation of MS-HRM use
 for age determination in long-lived species, such as Asian elephants. The
 average lifespan of Asian elephants is between 50-70 years but some have
 been known to survive for more than 80 years. DNA was extracted from 53
 blood samples of captive Asian elephants across 11 zoos in Japan, with
 known ages ranging from a few months to 65 years. Methylation rates of two
 candidate age-related epigenetic genes, RALYL and TET2, were significantly
 correlated with chronological age. Finally, we established a linear,
 unisex age estimation model with a mean absolute error (MAE) of 7.36
 years. This exploratory study suggests an avenue to further explore MS-HRM
 as an alternative method to estimate the chronological age of Asian
 elephants.
description: # Estimation of captive Asian elephants (Elephas maximus) age based on DNA
 methylation: An exploratory analysis using methylation-sensitive
 high-resolution melting (MS-HRM)
 ## Description of the data
 and file structure 1. The raw methylation data of *RALYL* and *TET2* used
 in this analysis are in csv files. This is the dataset that we used to
 develop the age estimation model. The '**subject ID**'
 represents the same individuals. In contrast, '**sample**'
 represents the sample collections taken over time. In addition, cells
 containing '**n/a**' in our dataset within the
 'sample' column are samples which were sampled recently and had
 no define number ID at the time (please refer to the Supplementary
 Information on the manuscript for more details on sampling date).
 '**sex**' represents the sex of the individual, where F: female
 and M: male. '**age**' represents the chronological age of the
 individual at the time of sampling.
 '**ralyl_methylationrate_ave**' and
 '**tet2_methylationrate_ave**' is the average methylation rate
 of two duplicate measure of each sample of *RALYL* and *TET2* regions
 respectively. '**ralyl_df_ave**' and '**tet2_df_ave**'
 is the Df value of two duplicate measure of each sample of *RALYL* and
 TET2 regions respectively. '**ralyl_before**',
 '**tet2_before**', and '**all_svm**' represents the
 predicted age output of the SVM model, before leave-one-individual-out
 cross-validation for the *RALYL* model, *TET2* model, and the combined
 model respectively. '**ralyl_loiocv_after**',
 '**tet2_loiocv**', and '**all_loiocv**' represents the
 output of the predicted age after leave-one-individual-out
 cross-validation for the *RALYL* model, *TET2* model, and the combined
 model respectively. 2. The R script is a pdf file. This is the R script
 for the statistical analysis and cross-validations examined in this study.
 The codes were created by modifying the R script of Qi et al. (2021)
 accordingly ().
 Cross-validation used to validate model performance was
 leave-one-individual-out cross-validation as samples from the same
 individuals were sampled over time.