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

ABSTRACT The use of long-read sequencing using portable Oxford Nanopore Technologies (ONT) 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 ONT sequencing of full-length 16S rRNA genes for diversity surveys of symbiotic dinoflagellate (family Symbiodiniaceae) microbiomes. When comparing amplicon sequencing of the full-length bacterial 16S rRNA gene with only its V4 hypervariable gene region using a known microbial community, the former recapitulated the bacterial taxonomic composition more accurately. ONT sequencing 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 ONT long-read sequencing as a powerful tool for marine microbiome studies to catalyze advancements in the fields of ecology and evolution, resource management, and conservation. Competing Interest Statement The authors have declared no competing interest. Footnotes Affiliation numbers were fixed to reflect correct numbering order https://github.com/VivianYifanLi/Li_et_al_2025_ONTSequencing