2025/03/17 by Emily V Kerns, Jesse N. Weber · 1 voice
Biochemistry, Genetics and Molecular Biology · Environmental Science · #Epigenetics and DNA Methylation #Environmental DNA in Biodiversity Studies #Cancer-related gene regulation
paper · pdf · doi:10.1101/2025.03.14.643302
openalex publication_date 2025/03/17 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/15
Abstract DNA methylation (DNAm) is the most commonly studied marker in ecological epigenetics, yet the performance of library preparation strategies and bioinformatic tools are seldom assessed in genetically variable natural populations. We profiled DNAm in threespine stickleback ( Gasterosteus aculeatus ) liver tissue, using reduced representation bisulfite sequencing (RRBS) and whole genome bisulfite sequencing (WGBS) across technical and biological replicates. We additionally collated publicly available RRBS and WGBS data from taxonomically diverse organisms, and then compared how the most commonly used methylation software (Bismark) performed relative to alternative pipelines (BWA meth, BiSulfite Bolt, and Biscuit). Even after choosing parameters to maximize Bismark’s mapping efficiency, it was still outperformed by all other methods. Surprisingly, newer tools overrepresented DNAm compared to older methods, highlighting the importance of testing methods on nonmodel organisms. There were also distinct differences in DNAm profiles produced across library preparation methods, with large impacts of population and read depth filters. Methylated sites unique to WGBS predominantly mapped to introns and intergenic regions, while sites unique to RRBS primarily overlapped with promoters and exons. Moreover, the prevalence of nucleotides with intermediate methylation (within individuals) was greatly reduced in RRBS. Together, this suggests that RRBS may be more useful for detecting functionally-relevant methylation differences. Based on these results, we provide methodological recommendations for improving the reliability and utility of DNAm profiles, particularly concerning the detection of functionally relevant DNAm differences in genetically diverse natural populations.