Introducing a novel 28S rRNA marker for improved assessment of coral reef biodiversity.

Biodiversity monitoring based on DNA metabarcoding depends on primer performance. Here, we develop a new metabarcoding primer pair that targets a ∼318 bp fragment of the 28S rRNA gene that is highly conserved across branches of the tree of life. We apply these novel primers to detect coral reef biodiversity and perform a direct comparison with the most commonly used metabarcoding marker for coral reefs, targeting the cyctochrome oxidase subunit I (COI) gene (DOI 10.1186/1742-9994-10-34). We show that our novel 28S primers detect a broader taxonomic scope of species across phyla and classes within complex coral reef communities than the Leray-Geller COI primers. We validate species detection, amplification efficiency, and the quantitative potential of these novel primers using a suite of mock and natural complex reef communities. In the natural complex coral reef community experiments, commonly used COI metabarcoding primers detected only 12 phyla (18 classes), whereas our new 28S primer increased detection to 16 phyla (19 classes). We further validate the performance of these new primers using sponges as the only taxonomic group in this natural community for which an exhaustive species-level metabarcoding reference database exists. Sponges are a notoriously challenging group for coral reef metabarcoding studies, and COI detected only 30.9% of the species identified by eye, whereas our primers increased that to 79.4% of species. Mock community experiments detected most of the sponge species present (94/146) spanning a broad taxonomic scope (15 orders), and showed limited taxon-specific primer biases with only a single species exceeding a two-fold deviation from the expected number of reads based on abundance. Limited primer bias allows for possible quantitative metabarcoding, and we further show a significant relationship between read abundance and percent coverage of the sponge taxa identified by eye (