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High-throughput sequencing is revolutionizing our ability to comprehensively characterize free-living and symbiotic Symbiodiniaceae, a diverse dinoflagellate group that plays a critical role in coral reef ecosystems. Most studies however, focus on a single marker for metabarcoding Symbiodiniaceae, potentially missing important ecological traits that a combination of markers may capture. In this proof-of-concept study, we used a small set of symbiotic giant clam ( Tridacna maxima ) samples obtained from nine French Polynesian locations and tested a dual-index sequence library preparation method that pools and simultaneously sequences multiple Symbiodiniaceae gene amplicons per sample for in-depth biodiversity assessments. The rationale for this approach was to allow the metabarcoding of multiple genes without extra costs associated with additional single amplicon dual indexing and library preparations. Our results showed that the technique effectively recovered very similar proportions of sequence reads and dominant Symbiodiniaceae clades among the three pooled gene amplicons investigated per sample, and captured varying levels of phylogenetic resolution enabling a more comprehensive assessment of the diversity present. The pooled Symbiodiniaceae multi-gene metabarcoding approach described here is readily scalable, offering considerable analytical cost savings while providing sufficient phylogenetic information and sequence coverage.

Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via metabarcoding of pooled multi-gene amplicons