Long-read Oxford Nanopore sequences and short-read Illumina MiSeq sequences of 16S rRNA from Symbiodiniaceae cultures and ZymoBIOMICS Microbial Community Standards

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. In this study, we assessed the reliability of long-read sequencing (ONT) to characterize microbiome compositions by comparing the results of full-length bacterial 16S rRNA sequencing (~1500 bp) with sequencing of the V4 hypervariable gene region (~350 bp) using a known microbial community as reference. In addition, we used ONT to survey six Symbiodiniaceae cultures harboring distinct bacterial microbiomes to assess consistency between sequencing runs and to determine optimal read depth. We further tested this method in the field to assess the impact of inevitable environmental contamination when working in minimally equipped field stations. Our results demonstrate that long-read ONT sequencing of the full-length 16S rRNA gene is highly accurate and consistent. It also achieves fine taxonomic resolutions with sufficient read depth to fully capture species richness. This work validates and informs future use of ONT for understudied microbiomes.