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

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.