Evaluation of Methods for Concentration and Analysis of Viral Sewage Metagenomes

Sewage is a valuable repository for tracking human pathogens in the environment. The detection and identification of viruses from sewage is crucial for wastewater-based epidemiology (WBE) and microbial source tracking (MST). This dissertation investigates the impacts of various concentration and processing methods on PCR and sequencing outcomes from the sewage virome. The goals of this research are (1) to observe how the concentration method impacts the recovery of different viruses, (2) to understand the presence and abundance of species within the sewage virome, and (3) to determine the feasibility of detecting pathogens via WBE and other innovative methods. The first study of this dissertation assesses the impacts of virus concentration method on the PCR recovery of common viral fecal indicators and pathogens and compares the native recovery to spike-in recovery. Amicon ultrafiltration resulted in the highest average PCR recoveries and the prefiltration step caused significant decreases in bacteria signal for all concentration methods. Spiked-in controls inconsistently reflected the concentration efficiency of the assayed targets, indicating that spike-ins should be chosen carefully to demonstrate recovery accurately. The next study explored the temporal fluctuations of the sewage virome over a short window to assess the impact of sampling frequency on target detection. Nanotrap particles were used to concentrate samples, dPCR was used to quantify viral fecal indicators and pathogens, and targeted sequencing was employed to help resolve low-titer pathogens from the bulk metagenome. Adenoviruses and astroviruses were the most prevalent species and were detected consistently throughout the sampling window, but multiple clinically-relevant targets were less abundant. It was determined that the frequency of sampling for WBE can significantly impact the recovery of low-titer targets. The following chapter assessed the impact of two concentration methods, Nanotrap particles and Amicon ultrafiltration, and observed geographic trends on sewage virome composition. Nanotrap-concentrated samples resulted in higher alpha diversities, and the concentration method was observed to have a greater impact on virome diversity than the geographic origin of the sample. Both concentration methods were effective for different pathogens, highlighting the need to optimize methods for specific targets. The final portion of this dissertation explores the use of Capillary Zone Electrophoresis (CZE) for separating viruses from mixed samples into distinct fractions. CZE was shown to effectively separate species into distinct migration windows using a curated phage mixture. Separating sewage into fractions increased the resolution of low-titer pathogens compared to a bulk metagenome.Overall, this work demonstrates the need for specific and targeted methods for processing wastewater for MST and WBE applications and contributes supporting data for multiple potential protocols.