VESPA: an optimized protocol for accurate metabarcoding-based characterization of vertebrate eukaryotic endosymbiont and parasite assemblages

Leah A. Owens (), Sagan Friant, Bruno Martorelli Di Genova, Laura J. Knoll, Monica Contreras, Oscar Noya-Alarcon, Maria G. Dominguez-Bello and Tony L. Goldberg ()
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Leah A. Owens: University of Wisconsin-Madison
Sagan Friant: University of Wisconsin-Madison
Bruno Martorelli Di Genova: University of Wisconsin-Madison
Laura J. Knoll: University of Wisconsin-Madison
Monica Contreras: Venezuelan Institute of Scientific Research (IVIC)
Oscar Noya-Alarcon: Centro Amazónico de Investigación y Control de Enfermedades Tropicales-CAICET
Maria G. Dominguez-Bello: Rutgers University–New Brunswick
Tony L. Goldberg: University of Wisconsin-Madison

Nature Communications, 2024, vol. 15, issue 1, 1-16

Abstract: Abstract Protocols for characterizing taxonomic assemblages by deep sequencing of short DNA barcode regions (metabarcoding) have revolutionized our understanding of microbial communities and are standardized for bacteria, archaea, and fungi. Unfortunately, comparable methods for host-associated eukaryotes have lagged due to technical challenges. Despite 54 published studies, issues remain with primer complementarity, off-target amplification, and lack of external validation. Here, we present VESPA (Vertebrate Eukaryotic endoSymbiont and Parasite Analysis) primers and optimized metabarcoding protocol for host-associated eukaryotic community analysis. Using in silico prediction, panel PCR, engineered mock community standards, and clinical samples, we demonstrate VESPA to be more effective at resolving host-associated eukaryotic assemblages than previously published methods and to minimize off-target amplification. When applied to human and non-human primate samples, VESPA enables reconstruction of host-associated eukaryotic endosymbiont communities more accurately and at finer taxonomic resolution than microscopy. VESPA has the potential to advance basic and translational science on vertebrate eukaryotic endosymbiont communities, similar to achievements made for bacterial, archaeal, and fungal microbiomes.

Date: 2024
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DOI: 10.1038/s41467-023-44521-3

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