A metagenomics approach to identification of eukaryotes in metazoan-associated microbiomes

Abstract

ABSTRACTBackgroundMicrobial eukaryotes are integral components of the microbiome where they shape community composition and ecological interactions. However, the abundance and diversity of eukaryotic species within the microbiome, the ‘eukaryome’, remains poorly defined. These deficiencies arise from unresolved technical limitations in recovering DNA from microbial eukaryotes due to their relatively low abundance in most samples and resilience to extraction. To overcome these limitations, we developed an extraction protocol that specifically targets recovery of eukaryotic microbes from microbiome samples and allows for metagenomics sequencing of eukaryotic species.MethodsMicrobes were seeded in synthetic stool prior to DNA extraction to mimic microbiome samples from the gastrointestinal tract. Assessment of cell disruption was performed using intracellular staining with the azo dye trypan blue or quantification of DNA recovery. A mock microbial community of five bacteria and five eukaryotes was built to test the effectiveness of the full protocol by seeding stool with defined numbers of cells from each species.ResultsMechanical disruption efficiently released DNA from bacterial, fungal, and protozoan species where standard microbiome DNA extraction kits did not. Optimization of the bead beating parameters lysed >95% of fungal cells within synthetic stool samples. In a mixed bacteria- eukaryote sample, eukaryotic DNA could be further enriched by targeting methylated DNA for destruction with methyl-specific restriction endonucleases. Application of this approach to a defined community of 10 different microbes, five eukaryotes and five bacteria, seeded in synthetic stool demonstrated the success of this strategy by enriching for eukaryotes approximately 72-fold and producing a eukaryote-dominated DNA pool.ConclusionsOverall, development of a microbiome sample protocol that includes DNA extraction and enrichment from eukaryotic species will facilitate exploration of the eukaryome and its impact on human health.