Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

Recirculating aquaculture systems (RAS) heavily depend on microbial communities to maintain water quality. These communities therefore influence the growth, development, and welfare of farmed fish. With the increasing socio-economic role of fish farming e.g. regarding food security, an in-depth understanding of aquaculture microbial communities is also relevant from a management perspective. However, the data situation regarding the composition of microbial communities within RAS is patchy. Since this is partly ascribed to method choices, there clearly is a need for accurate, standardized, and user-friendly methods to study microbial communities in aquaculture systems. Here, we compare the performance of 16S amplicon sequencing, Pac-Bio long-read amplicon sequencing, and amplification-free shotgun metagenomics in the characterization of microbial communities in two commercial-size RAS fish farms. We show that, even though primer choice affects read quality, diversity, and assigned taxa, distinct primer pairs uncover similar spatio-temporal patterns between sample types, farms, and time points. We find that long-read amplicons underperform regarding quantitative resolution of spatio-temporal patterns, but allow for species-level identification of functional services and pathogens. Finally, shotgun metagenomics data identified fungi, viruses, and bacteriophages, opening avenues for an exploration of natural approaches regarding antipathogenic treatments. Overall, the datasets agreed on major prokaryotic players. In conclusion, different sequencing approaches yield overlapping and highly complementary results, with each contributing data no other approach could. Such a tiered approach therefore constitutes a practical and cost-effective strategy for obtaining the maximum amount of information on aquaculture microbial communities. These data could lead to better farm management practices and at the same time inform basic research on community evolution dynamics.