Comparison of high‐throughput sequencing methods for bacterial microbiota profiling in catfish aquaculture

Abstract

AbstractObjectiveBacterial community profiling in aquaculture can be useful for monitoring environmental conditions that may lead to disease outbreaks, understanding the impact of dietary treatments on fish health, and identifying microbes that have potential as probiotics. Microbiota characterization is commonly performed with high‐throughput sequencing using short‐read platforms (i.e., Illumina) targeting variable regions of the bacterial 16S ribosomal RNA (rRNA) gene. Long‐read platforms (i.e., Oxford Nanopore Technologies [ONT]) enable sequencing of full‐length 16S rRNA genes. While short‐read platforms are highly accurate, long‐read platforms sacrifice sequence quality in exchange for longer read lengths.MethodsHerein, a typical short‐read approach (Illumina MiSeq; V4 region of 16S rRNA gene) was compared with two near‐full‐length 16S rRNA gene ONT protocols (ONT Custom and ONT 16S). Sequencing was performed on a mock community composed of fish‐relevant bacteria, in addition to samples (digesta, gill, skin) obtained from 10 hybrid catfish (female Channel Catfish Ictalurus punctatus × male Blue Catfish I. furcatus) and their environment (pond and holding tank). Fish were euthanized with either an overdose of MS‐222 or cranial concussion and subsequent pithing to enable comparisons of these sequencing protocols in the context of a realistic experimental design.ResultThe ONT Custom protocol best recapitulated the theoretical composition of the mock community, followed by the ONT 16S and Illumina MiSeq protocols. Phylum‐level composition was consistent within sample type across sequencing methods. However, at lower levels there were biases for several taxa, particularly in pond samples. Euthanasia method also appeared to introduce differences, primarily on the cutaneous communities. The three sequencing methods consistently identified significant differences between euthanasia methods, with Illumina MiSeq revealing the greatest differences. While all strategies performed comparably for digesta, skin, and water samples, the ONT strategies underperformed for gill samples.ConclusionDepending on application, results indicate that near‐full‐length 16S rRNA gene nanopore sequencing is a viable option for aquaculture microbiota studies.