DOI: 10.5061/dryad.wstqjq2wq
title: Independent avian epigenetic clocks for aging and development

description: Information on individual age is a fundamental aspect in many ecological
 and evolutionary studies. However, accurate and non-lethal methods that
 can be applied to estimate the age of wild animals are often absent.
 Furthermore, since the process of aging is accompanied by a physical
 decline and the deterioration of biological functions, the biological age
 often deviates from the chronological age. Epigenetic marks are widely
 suggested to be associated with this age-related physical decline, and
 especially changes in DNA methylation are suggested to be reliable
 age-predictive biomarkers. Here, we developed separate epigenetic clocks
 for aging and development in a small passerine bird, the great tit (Parus
 major). The aging clock was constructed and evaluated using erythrocyte
 DNA methylation data of 122 post-fledging individuals, and the
 developmental clock using 67 pre-fledging individuals from a wild
 population. Using a leave-one-out cross validation approach, we were able
 to accurately predict the ages of individuals with median absolute
 deviations of 0.40 years for the aging and 1.06 days for the development
 clock. Moreover, using existing data from a brood-size manipulation we
 show that nestlings from reduced broods are estimated to be biologically
 older compared to control nestlings, while they are expected to have
 higher fitness. These epigenetic clocks provide further evidence that, as
 observed in mammals, changes in DNA methylation of certain CpG sites are
 highly correlated with chronological age in birds and this opens up new
 avenues for broad applications in behavioural and evolutionary ecology.
description: # Independent avian epigenetic clocks for aging and development
 **Description of data and file structure** *
  "barcode_files_epiGBS2_2018_B1-B8",
 "barcode_files_epiGBS2_2018_B9-B10",
 "barcode_files_epiGBS2_2019_B13-B18" &
 "barcode_files_epiGBS2_2020_B19-B23" includes the unique barcode
 combinations needed to demultiplex the raw DNA methylation (epiGBS2) data
 on NCBI (see below). Column name explanation: Flowcell = sequencing
 flowcell; Lane = sequencing lane; Barcode_R1 = barcode ligated to the 5
 prime – 3 prime DNA strands (i.e. R1 or BA); Barcode_R2 = barcode ligated
 to the 3 prime – 5 prime DNA strands (i.e. R2 or CO); Sample = individual;
 ENZ_R1 = restriction enzyme; ENZ_R2 = restriction enzyme; Wobble_R1 =
 length (in nucleotides) of the random nucleotide sequence (i.e. UMI) in
 the adapter of the 5 prime – 3 prime DNA strands; Wobble_R2 = length (in
 nucleotides) of the random nucleotide sequence (i.e. UMI) in the adapter
 of the 3 prime – 5 prime DNA strands; Species = study species. The year in
 the file name stands for the year of the sample collection (i.e.
 experimental study). -  "Extern_Samples_DevelopClock.txt"
 includes the information on the pre-fledging samples. Column Explanation:
 Sample_ID = contains unique IDs for eachs sample, RNR = contains unique ID
 for each individual, Age_in_Days = Chronological age in Days, Mol_sex =
 sex of the pre-fledgings, Experiment = experiment they were used in,
 Treatment = the treatment within each experiment, Filename = the
 methylation call file name, nr_cpg = If the file contained more or less
 than 3m unique rows, Genetic_ID = the brood of origin of individuals from
 a brood size manipulation experiment. *
 "Extern_MethData_Train_DevelopClock.txt" includes the Sample_IDs
 of pre-fledging samples and the corresponding methylation ratios of 11
 thousand CpGs that were used to train the Developmental Clock. Column
 Explanation: Sample_ID = contain unique ID for eachs sample, the remaining
 columns represent different CpG sites. -
 "Extern_MethData_Brood_Test_DevelopClock.txt" includes the
 methylation ratio of the 115 CpGs that were used in the final
 developmental clock for 102 samples that were part of the brood size
 manipulation experiment. Column Explanation: each column represent an
 unique ID for each sample, each row represent one of the 115 CpGs. *
 "Extern_Samples_AgeingClock.txt" includes the information of the
 post-fledging samples. Column Explanation: RNR = unique ID for each
 individual, Age_in_Days = Chronological age in Days, Mol_sex = sex of the
 post-fledgings, Filename = the methylation call file name. -
 "MethylationData_AgeingClock.txt" includes the methylation
 ratios of 35 thousand CpG sites for 122 post-fledging individuals that was
 used to train the Ageing Clock. Column Explanation: CpG = the name of each
 unique CpG, the remaining columns represent the unique IDs for each
 individual with the corresponding methylation ratio. Any cells that
 contain "NA" represent missing values or the column does not
 apply to that individual. * "MethylationCalling_DevelopClock.R"
 includes the script to filter the DNA methylation (epiGBS2) data for the
 pre-fledgings, results in
 "Extern_MethData_Train_DevelopClock.txt" datasheet that was used
 to train the Development Clock model. -
 "MethylationCalling_AgeingClock.R" includes the script to fiter
 the DNA methylation (epiGBS2) data for the post-fledgings, results in
 "MethylationData_AgeingClock.txt" datasheet that was used to
 train the Ageing Clock model. * "Extern_Clock_Analysis.pdf"
 contains the codes used to train and test both the epigenetic clock
 models. - The raw genomic epiGBS2 reads are available on NCBI under
 BioProject PRJNA208335 under the SRA accessions SRX18523280, SRX18523281,
 SRX18523282, SRX18523283, SRX18523284, SRX18523285, SRX18523286,
 SRX18523287, SRX21758799, SRX21758800, SRX22027777, SRX22027778,
 SRX22027779, SRX22027780, SRX22027781, SRX26112229, SRX26112230,
 SRX26112231, SRX26112232, SRX26112233 and SRX26112234. For more
 information on data collection and analysis, please refer to the published
 manuscript and the Supplementary Material. Also, please feel free to
 contact Kees van Oers
 ([k.vanoers@nioo.knaw.nl](mailto:k.vanoers@nioo.knaw.nl)) if you have any
 further questions or comments. The epiGBS2 bioinformatics pipeline can be
 accessed on github
 ([https://github.com/nioo-knaw/epiGBS2](https://github.com/nioo-knaw/epiGBS2)).