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.

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
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.

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.