Assessment of next generation amplicon sequencing of the beta-giardin gene for the detection of Giardia duodenalis assemblages and mixed infections

Giardia duodenalis is an enteric protozoan parasite commonly found in humans and many other animals around the world. The parasite is grouped into genetically related strains called assemblages which display differing degrees of host specificity. Although mixed assemblage infections have been documented the full extent of the occurrence and importance of mixed infections remains to be characterized as current sequencing technologies lack the sensitivity to readily detect mixed infections. Here we have developed a next generation amplicon sequencing (NGS) protocol and analysis pipeline for detecting Giardia assemblages using the beta-giardin gene. NGS was validated using 37 isolates that included Giardia muris and six assemblages (A-F) of Giardia duodenalis obtained from seven different hosts. NGS was compared to traditional PCR and direct Sanger sequencing for its ability to detect Giardia species, assemblages, and mixed assemblage infections. We demonstrate that NGS works as well as PCR and Sanger sequencing for assemblage detection as the same assemblage was observed in all samples by both methods. NGS has the further benefit of detecting mixed assemblage infections, low abundance assemblages, and intra-assemblage variation in samples which would have been missed using direct Sanger sequencing alone. NGS represents a powerful new tool for exploring Giardia infections not only in infected hosts but also in environmental specimens which may aide in understanding Giardia epidemiology.