Most of the advances in microbiology over the past 15 years have focused on bacteria and, to a lesser extent, on archaea and viruses. Protists (microbial eukaryotes), on the other hand, are relatively unstudied, in part because their genomes are large, complex, and poorly represented in the reference genome collections. Funds from this grant support work by Professor Jane Carlton, a leading protist metagenomic expert, to conduct a pilot project to discover protists in pets and pests in all five boroughs of New York City. Carlton will team up with researchers at Fordham University, Barnard College, Hunter College, and the Department of Environmental Protection to collect samples from 20 cats, 20 dogs, 20 rats, 20 mice, 20 cockroaches, and 20 pigeons from each of the five boroughs of New York City, for a total of 600 samples. The team will then use wet-lab methods and computational pipelines to characterize protists found in sewage collected from 14 NYC treatment plants, which service the five NYC boroughs. These data will then be used to amplify and characterize the 18S rRNA marker gene from the pet and pest samples to characterize community diversity and look for associations between the protists found in sewage and the pets and pests that harbor them. The overarching goal is to develop and demonstrate the viability of methods to reliably discover protists in host organisms.