Fish microbiomics: Strengths and limitations of MinION sequencing of gilthead sea bream (Sparus aurata) intestinal microbiota

There are several affordable methods involving different sequencing technologies for microbial characterization. However, the choice of the sequencing platform and the downstream analysis can yield somewhat different results. Here we aimed to examine the strengths and limitations of different sequencing platforms for their use in aquacultured fish gut mucosal samples, using gilthead sea bream (Sparus aurata) as a model. The attention was focused on the portable Oxford Nanopore Technologies (ONT) MinION device, which offers the possibility of fast profiling of mucosal microbial samples. The standard PCR protocol provided for the MinION full-length sequencing of the 16S rRNA was optimized (input material, annealing temperature and number of cycles) for fish mucosal samples and the optimized protocol was validated using a standard mock community with known bacterial taxa and abundances. The optimized PCR (annealing temperature 52 °C, 30 cycles) yielded highly accurate results when sequencing mock community samples (R2 = 0.95) and was chosen for the subsequent analyses. Finally, the sequencing results were compared to those from Illumina MiSeq sequencing of the V3-V4 region of the 16S rRNA gene to determine strengths and weaknesses from both platforms. Our results showed that MinION is a reliable and accurate tool for the assessment of intestinal bacteria communities, yielding similar results to Illumina with correlation coefficients >0.75. However, biologically important but less abundant taxa (e.g. Actinobacteriota and Bacteroidota) were apparently masked when comparisons were made with the Illumina MiSeq results. We conclude that the choice of sequencing platform depends on the type of sample and scientific question. Thus, when evaluating fish gut mucosal samples, where the biological interpretation focuses on taxa related to gut function and metabolism, Illumina MiSeq allows a broader amplification of taxa of interest, while MinION provides good results in terms of abundance and fast profiling of microbial communities, making it very attractive for studies focused on environmentally-related samples (e.g. gills and skin samples).This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 871108 (AQUAEXCEL3.0). This output reflects only the author's view and the European Commission cannot be held responsible for any use that may be made of the information contained therein. MCP was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018–024049-I co-funded by the European Social Fund (ESF) & ACOND/2022 Generalitat Valenciana). STR was funded by a Marie Skłodowska-Curie Innovative Training Network (ITN) fellowship under grant agreement No. 956697 (EATFISH)