Abstract
DNA methylation in CpG dinucleotides is an important epigenetic mark in fish spermatozoa since it has been shown that some sperm methylome features are transmitted to the offspring. Reduced representation bisulfite sequencing (RRBS) is one genome-scale methods developed to assess DNA methylation at CpG sites. It allows the sequencing of a reduced fraction of the genome expected to be enriched in CpGs. The aim of this study is to characterize the extent of the CpG sites that can be identified in the RRBS-reduced sequenced fraction of rainbow trout spermatozoa, in order to evaluate the potential of RRBS for sperm DNA methylation studies. We observed that RRBS did provide a reduced amount of genomic data, the sum of the CpGs analyzed on 12 males spanning 9% of the total genomic CpGs. CpGs were only slightly enriched in the RRBS data (×1.7 times the sequenced nucleotides), the possible causes being linked to trout genome structure and sequenced fragments size. All genomic functional features were represented in our CpG dataset, with a noticeable enrichment in exons but, strikingly, not in promoters. The number of CpGs shared between biological replicates was low, but this proportion reached workable values from six biological replicates (46% of the analyzed cytosines) on. The choices that are to be made regarding fragment size selection and the options during bioinformatic data processing are discussed. In all, RRBS is a relevant first-approach method to scan the CpG DNA methylation status of spermatozoa along rainbow trout genome, although in a very reduced pattern among biological replicates.
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Data availability
The data for this study has been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB60579 (https://www.ebi.ac.uk/ena/browser/view/PRJEB60579).
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Acknowledgements
The authors acknowledge the skillful involvement of the staff from the ISC LPGP (Infrastructure Scientifique Collective du Laboratoire de Physiologie et Génomique des Poissons) and UE INRAE PEIMA for animal rearing and care. The authors wish to warmly thank Vincent Coustham for his valuable comments on the interpretation of our RRBS data which triggered the motivation to write this paper. We are grateful to the Genotoul bioinformatics platform Toulouse Occitanie (Bioinfo Genotoul, doi: 10.15454/1.5572369328961167E12) for providing computing and storage resources.
Funding
MEK is recipient of a PhD fellowship ARED from region Bretagne and INRAE PHASE (2020–2023). AS obtained the financial support from the internal funding scheme at the Norwegian University of Life Sciences (project number 1211130114), which financed AS international stay at INRAE, Fish Physiology and Genomics, UR 1037, Rennes, France. This work was funded by the French CRB Anim project ANR-11-INBS-0003 and the European FEAMP BIOGERM measure 47 (Innovation Aquaculture).
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MEK organized and carried out the study, analyzed and interpreted the data, and drafted the manuscript. AB taught and supervised the bioinformatic work of MEK, and contributed to most bio-informatic analyses. AS contributed to the wet lab work. DL contributed to the conception of the project and provided her knowledge of the trout genome for data interpretation. AL and CL conceived, designed and supervised the study. CL supervised the writing of the manuscript and AL provided her knowledge on genomic DNA methylation in trout. All authors approved the final version of the manuscript.
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Rainbow trout males were kept in the experimental fish facilities ISC LPGP (Infrastructure Scientifique Collective du Laboratoire de Physiologie et Génomique des Poissons) of INRAE (Agreement number D-35-238-6) with full approval for experimental fish rearing in strict accordance with French and European Union Directive 2010/63/EU for animal experimentation. The Institutional Animal Care and Use Ethical Committee in Rennes LPGP (Fish Physiology and Genomics Department) specifically approved this study (no. T-2020-37-CL). All fish were handled for gamete collection in strict accordance with the guidelines of the Institutional Animal Care and Use Ethical Committee in Rennes LPGP. Catherine Labbé is accredited by the French Veterinary Authority for fish experimentation (no. 005239). The animal study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for animal research.
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El Kamouh, M., Brionne, A., Sayyari, A. et al. Strengths and limitations of reduced representation bisulfite sequencing (RRBS) in the perspective of DNA methylation analysis in fish: a case-study on rainbow trout spermatozoa. Fish Physiol Biochem (2024). https://doi.org/10.1007/s10695-024-01326-5
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DOI: https://doi.org/10.1007/s10695-024-01326-5