Improved characterisation of coral-associated fungal communities using host DNA depletion and a novel ITS primer

Fungi are increasingly recognised as integral components of the coral holobiont, with predicted roles in nitrogen cycling, nutrient acquisition and promoting stress tolerance. However, characterising coral-associated fungal communities using metabarcoding remains challenging due to extensive co-amplification of host DNA, often leading to low fungal read recovery and an underestimation of diversity. Here, we developed and validated a robust fungal metabarcoding protocol that consistently improved fungal reads across coral species for comprehensive diversity analyses. We addressed host co-amplification through two approaches: (i) host depletion with the Zymo HostZERO Microbial DNA kit and (ii) a newly designed forward primer, ITS3-CoralF, with mismatches to coral DNA to reduce their amplification when paired with the ITS4 reverse primer. We assessed the extraction kit and primer biases and performance across reef sediment samples, and the tissue and skeletal compartments of three species of corals, Pocillopora acuta, Pachyseris speciosa, and Diploastrea heliopora. The HostZERO kit consistently reduced non-target eukaryotic DNA across sample types, enhancing the detection of rare fungi despite shifts in community composition. In coral tissues, ITS3-CoralF achieved at least an 800-fold increase in fungal reads compared to universal primers, though with some bias against rare taxa. For skeletons, thorough removal of tissue prior to total DNA extraction was sufficient, though ITS3-CoralF further improved fungal recovery. Together, our results provide a validated pipeline for profiling fungi across coral hosts, allowing improved recovery of fungal diversity and a deeper understanding of the coral mycobiome.