Evaluation of full-length 16S rRNA amplicon sequencing using Oxford Nanopore Technologies for diversity surveys of understudied microbiomes

The use of long-read sequencing is becoming increasingly popular in the study of host-associated microbiomes. However, its application has not yet been optimized for characterizing understudied microbiomes, such as those in marine environments. We evaluated the accuracy and consistency of long-read sequencing of full-length 16S rRNA genes for diversity surveys of symbiotic dinoflagellate (family Symbiodiniaceae) microbiomes. When comparing long-read amplicon sequencing of the full-length bacterial 16S rRNA gene with short-read sequencing of the V4 hypervariable gene region using a known microbial community, the former recapitulated the bacterial taxonomic composition more accurately. Long-read sequencing performed using Oxford Nanopore Technologies was also highly consistent between sequencing runs and flow cells. Long-read sequencing technologies enable microbiome surveys using the full-length 16S rRNA gene, achieving higher accuracy and resolution. This work validates long-read sequencing as a robust approach for resolving microbiome composition in understudied marine systems, thereby supporting ecological and evolutionary investigations of host–microbe associations.