Abstract
Coral reefs are ecosystem engineers by providing complex habitat structures that promote prey refuge and marine life diversity. However, the existence of coral reefs would be impossible without their complex symbiotic connections with their microbial partners. The symbiotic consortia between the coral and their microorganisms constitute the coral holobiont. Over the past decade, second-generation short-read sequencing (typical length of 300 to 600 base pairs) metabarcoding has facilitated the identification and taxonomic classification of coral-associated microbes with some confidence to the genus level. However, this approach has not achieved lower taxonomic resolution (species or lower), resulting in low-resolution microbial identification. By utilizing ONT third-generation long-read sequencing coupled with universal prokaryotic and eukaryotic long-read primers that recover the complete rRNA operon spanning around 4,500 bp, we aim to enhance the resolution of the taxonomic classification. Coral samples were taken in triplicate from three species (Pseudodiploria strigosa, Pseudodiploria clivosa, and Orbicella faveolata) and obtained from shallow reefs off the coast of Miami, Florida. Sequencing the prokaryotic and eukaryotic communities of the corals was completed using ONT MinION for long-reads and Illumina MiSeq for short-read comparisons. We compared Illumina methods to novel ONT methodologies using a modified version of RESCUE, a previously published long-read ONT-based bioinformatics pipeline. Alpha and beta diversity, as well as community composition metrics, were evaluated to determine the differences between both approaches. Based on the results, we can conclude that short-read sequencing is more reliable than long-read sequencing because long reads have a relatively higher error rate and the lack of suitable reference databases relative to short-read sequencing. With a well-curated ribosomal operon database and better chemistry, ONT sequencing could potentially be a powerful high-throughput technique that researchers can use globally.