DOI: 10.5061/dryad.m0cfxpnz4
title: Data from: A clockwork fish. Age-prediction using DNA methylation-based biomarkers in the European seabass

description: Age-related changes in DNA methylation do occur. Taking advantage of this,
 mammalian and avian epigenetic clocks have been constructed to predict
 age. In fish, studies on age-related DNA methylation changes are scarce
 and no epigenetic clocks are available. However, in fisheries and
 population studies there is a need for accurate estimation of age,
 something that is often impossible for some economically important species
 with the currently available methods. Here, we used the European sea bass,
 a marine fish where age can be known with accuracy, to construct a piscine
 epigenetic clock, the first one in a cold-blooded vertebrate. We used
 targeted bisulfite sequencing to amplify 48 CpGs from four genes in muscle
 samples and applied penalized regressions to predict age. We, thus,
 developed an age predictor in fish that is highly accurate (0.824) and
 precise (2.149 years of error). In juvenile fish, accelerated growth due
 to elevated temperatures had no effect in age prediction, indicating that
 the clock is able to predict the chronological age independently of
 environmentally-driven perturbations. An epigenetic clock developed using
 muscle samples accurately predicted age in samples of testis but not
 ovaries, possibly reflecting the reproductive biology of fish. In
 conclusion, we report the development of the first piscine epigenetic
 clock, paving the way for similar studies in other species. Piscine
 epigenetic clocks should be of great utility for fisheries management and
 conservation purposes, where age determination is of crucial importance.
description: Overall design: We used multiplexed bisulfite sequencing (MBS) to
 measure the DNA methylation of specific regions in tissues of sea bass
 from different age classes. Two MBS libraries were prepared and sequenced.
 In all cases, each tissue and age class were represented by 4 replicate
 fish. The first MBS library consisted of 22 valid amplicons amplified in
 ovary, testis and muscle of 1.28, 3.07 and 10.5 years sea bass. The second
 MBS library consisted of 4 valid amplicons amplified in muscle of a) 0.55,
 0.96, 1, 1.1, 1.37, 1.64, 4.17, 5.83 and 6.75 years old sea bass reared at
 natural (low-17ºC) temperature and b) 0.48, 0.55, 0.82, 0.96, 1.1, 1.37
 years old sea bass reared at high (21ºC) temperature from 7 to 68 days
 post fertilization. Description of protocols: Fish were
 raised at the Aquarium facilities of the Institute of Marine Sciences or
 the Institute of Aquaculture Torre de La Sal (Spanish National Research
 Council, CSIC) following standard procedures until sampling. Samples were
 snap frozen in liquid nitrogen and kept at -80ºC until DNA extraction.
 Genomic DNA was extracted by the Phenol/Chlorofom/Isoamyl-alcohol
 (25:24:1) extraction protocol. MBS libraries were prepared as described in
 Anastasiadi et al 2018 (https://doi.org/10.1080/15592294.2018.1529504).
 Briefly, 2 μg of DNA were bisulfite-converted and PCRs with primers
 targeting specific bisulfite-converted regions were performed. Bead-based
 normalization of DNA quantities was followed by pooling of amplicons per
 sample. Indices were added to samples following a dual-index strategy by
 PCRs. Equal quantities of samples were pooled into a single final library
 and sequenced in an Illumina MiSeq using the 300 bp paired-end
 protocol. Description of data processing: Raw reads
 were quality trimmed by the Trim Galore for MBS 1 and Trimmomatic for MBS
 2<br> Trimmed reads were aligned to the reference genome dicLab
 (v1.0c, June 2012; http://seabass.mpipz.mpg.de/) using Bismark with
 --non_directional --phred33-quals --score_min L,0,-0.6. For samples of
 1.28, 3.07 and 10.5 years alignments were performed in three steps: 1)
 paired reads were aligned, 2) unmapped reads from the first step were
 aligned as single reads, and 3) unpaired reads from the first step of
 trimming were aligned like the unmapped reads. Paired-end and single-end
 alignments are provided as .bam files. Methylation calling was performed
 by the bismark_methylation_extractor. For samples of 1.28, 3.07 and 10.5
 years extraction was performed separately for paired-end and for
 single-end reads. Methylation files were merged into a single file
 containing all samples. Methylation values were read into R. CpGs with
 less than 5 coverage were eliminated. In this data
 package, two types of data are included per sample: alignmnet files (.bam)
 and methylation value of each CpG (.txt). For the MBS1, paired-end and
 single-end alignments (.bam) are provided. The processed data files are
 tab-delimited and contain: 1) the name of the sample, 2) the genomic
 position of the CpG (in the format chr, cpg start.cpg end), 3) the percent
 methylation and 4) the age in years.
description: A spreadsheet is included in this data package. It includes as
 columns the name of each sample, the age, tissue, developmental
 temperature, sex and the names of the corresponding alignment (for MBS1,
 both paired-end and single-end) and methylation values files.