Crouch, A. L.; Monsey, L.; Ramos, C.; Yracheta, J.; Anderson, M. Z.

Microbes in the microbiome form complex and dynamic microbial communities in and on their host where they are increasingly associated with host disease and behavior. While these microbial communities include bacterial, archaeal, viral, and eukaryotic constituents, most studies have focused on bacteria due to their dominance in the human host and associated technical limitations with recovering less abundant microbial kingdoms. Accumulating evidence suggests microbial eukaryotes in the microbiome play pivotal roles in host health, but our understandings of these interactions is limited to a few readily identifiable taxa. Here, combined cell sorting, optimized eukaryotic cell lysis, and unbiased sequencing to target host-associated microbial eukaryotes for metagenomic discovery and analysis. Using synthetic microbial communities, the optimized lysis and DNA recovery method for eukaryotes alone produced a DNA pool containing 38% eukaryotic DNA from a 1% microbial eukaryote population. Cell sorting on physiological properties of eukaryotic cells increased the number of metagenomic reads from microbial eukaryotes by up to 28-fold compared to commercial kits in human stool samples. Read frequencies increased by 10,000x on average for fungi identified in the Human Microbiome Project through metagenomic sequencing and allowed for the identification of novel taxa, de novo assembly of contigs from previously unknown microbial eukaryotes, and gene prediction from recovered genomic segments. These advances pave the way for the unbiased inclusion of microbial eukaryotes in deciphering determinants of health and disease in the host-associated gut microbiome.