Assessment of the effectiveness of host depletion techniques for profiling fish skin microbiomes and metagenomic analysis

Bell, Ashley G., Cable, Jo ORCID: https://orcid.org/0000-0002-8510-7055, Temperton, Ben and Tyler, Charles R. 2025. Assessment of the effectiveness of host depletion techniques for profiling fish skin microbiomes and metagenomic analysis. Microbiology Spectrum , e01838-25. 10.1128/spectrum.01838-25

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Abstract

Microbiomes on fish mucosal surfaces play crucial roles in nutrient absorption, immune priming, and defense, and disruptions in these microbial communities can lead to adverse health outcomes, including disease. Studying fish microbiomes relies on sequencing microbiota within mucosal-rich samples; however, nucleic acid extraction from these samples is composed predominantly of host DNA, making subsequent bioinformatic processes difficult. Host depletion techniques address this issue by either selectively degrading host DNA before sequencing or retaining bacterial DNA post-extraction. However, their application to fish mucosal samples has been largely unexplored. Here, we assessed the efficacy of various host depletion techniques on fish skin mucosal swabs via either selectively removing CpG-methylated (predominantly eukaryotic) DNA or selectively lysing eukaryotic cells before DNA extraction. Surprisingly, none of the existing methods we assessed effectively reduced host DNA to be practically useful. Furthermore, some methods introduced a bias toward certain bacterial taxa, including the Bacilli class and the Proteobacteria phylum. Our findings illustrate that the currently available host depletion techniques are largely ineffective for reducing host DNA in fish mucosal samples. This poses a major limitation for developing an understanding of the functional composition of fish mucosal microbiomes, as enriching microbiota (and excluding host DNA) is fundamental for cost-effective metagenomic studies and facilitating more accurate analyses of the microbiota metabolome and proteome. IMPORTANCE Microbial communities on fish mucosal surfaces are vital for immune function and disease resistance. However, sequencing these communities is hindered by the dominance of host DNA in mucosal samples, which can exceed 99% of total nucleic acids. While host depletion techniques are routinely used in human and mammalian systems to enrich microbial DNA, their efficacy on fish samples remains uncharacterized. In this study, we assessed multiple commercial and published host depletion methods on fish skin microbiomes. None significantly reduced host DNA to levels suitable for high-quality metagenomic sequencing, and some introduced taxonomic bias. We suggest methodological reasons, including differences in fish cell structure and mucus composition compared to mammalian systems, that may explain these shortcomings. Based on our findings, we propose protocol modifications and highlight key areas for improvement. This work identifies critical limitations and offers a foundation for developing optimized host depletion strategies tailored to fish mucosal microbiome research.

Item Type:	Article
Date Type:	Published Online
Status:	In Press
Schools:	Schools > Biosciences
Additional Information:	License information from Publisher: LICENSE 1: URL: https://creativecommons.org/licenses/by/4.0/, Start Date: 2025-12-22
Publisher:	American Society for Microbiology
Date of First Compliant Deposit:	7 January 2026
Date of Acceptance:	24 November 2025
Last Modified:	07 Jan 2026 12:00
URI:	https://orca.cardiff.ac.uk/id/eprint/183655

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