Moving Beyond Oxford Nanopore Standard Procedures: New Insights from Water and Multiple Fish Microbiomes

Oxford Nanopore Technology (ONT) allows for the rapid profiling of aquaculture microbiomes. However, not all the experimental and downstream methodological possibilities have been benchmarked. Here, we aimed to offer novel insights into the use of different library preparation methods (standard-RAP and native barcoding-LIG), primers (V3-V4, V1-V3, and V1-V9), and basecalling models (fast-FAST, high-HAC, and super-accuracy-SUP) implemented in ONT to elucidate the microbiota associated with the aquatic environment and farmed fish, including faeces, skin, and intestinal mucus. Microbial DNA from water and faeces samples could be amplified regardless of the library-primer strategy, but only with LIG and V1-V3/V1-V9 primers in the case of skin and intestine mucus. Low taxonomic assignment levels were favoured by the use of full-length V1-V9 primers, though in silico hybridisation revealed a lower number of potential matching sequences in the SILVA database, especially evident with the increase in Actinobacteriota in real datasets. SUP execution allowed for a higher median Phred quality (24) than FAST (11) and HAC (17), but its execution time (6-8 h) was higher in comparison to the other models (0.6-7 h). Altogether, we optimised the use of ONT for water- and fish-related microbial analyses, validating, for the first time, the use of the LIG strategy. We consider that LIG-V1-V9-HAC is the optimal time/cost-effective option to amplify the microbial DNA from environmental samples. However, the use of V1-V3 could help to maximise the dataset microbiome diversity, representing an alternative when long amplicon sequences become compromised by microbial DNA quality and/or high host DNA loads interfere with the PCR amplification/sequencing procedures, especially in the case of gut mucus.