Primary Contacts: Anita Wray, anita.wray@noaa.gov, Marty Kardos, martin.kardos@noaa.gov
We leveraged findings from our ongoing research to improve estimates of age for both vermilion and sunset rockfish. Specifically, we will use epigenetic analyses of fin clips to estimate the age of individuals in both species using a subset of our existing 30,000 vermilion and sunset rockfish samples collected from 2004-2023 during two industry-collaborative research surveys. Epigenetic ageing provides a non-lethal method of determining age and lifespan using the degree of methylation at genomic sites. These sites are preselected from previous studies.
We used 96 samples from a wide age distribution and good DNA quality from the fin clip. These samples were both Vermilion and Sunset rockfish from both the H&L and Trawl surveys.
Used the previously published genome of vermilion rockfish to identify the location of informative methylation markers used in European Bass, Australian lungfish, zebrafish, Murray cod, and Mary River cod (Anastasiadi & Piferrer, 2019, Mayne et al. 2021, Mayne et al. 2022). We did this with two methods: 1. Use LASTZ to align the vermilion and zebrafish genomes then filtered to only unique sites (see bash and R script) 2. Subsetted the zebrafish genome to only 300bp surrounding the CpG sites (600bp total, fasta file included in repo) then used NCBI BLAST against the vermilion genome
Developed primers to target those specific regions of the genome. We used methprimer to develop primers based on the LASTZ and BLAST outputs. This resulted in the .csv file which gave us primers, annealing temps, and amplicon lengths.
Followed protocols outlined in Mayne et al. 2021, Mayne et al. 2022, and Anastasiadi & Piferrer, 2019 to extract, bisulfite treat the DNA and amplify, barcode and sequence specific regions in the genome. (NOT INCLUDED IN THIS REPO)
Demultiplex, align, and call methylation percentage for each locus amplified. Most of the scripts are taken from this site, which was super helpful. In order, you trim, then align, then call the methylation percentage. The file that we end up using for step 5 is the .cov file from each individual, produced from the bismark_part1.sh script.
Ran a GLM in R to estimate age using methylation % at each locus
Our results suggest that although our loci are somewhat informative of age we do not have enough loci to have a confident age call for individuals. In order to increase confidence, we would need to locate, amplify, and sequence more CpG sites.
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