A comparative study of flow cytometry‐sorted communities and shotgun viral metagenomics in a Singapore municipal wastewater treatment plant

Abstract

Traditional or "bulk" viral enrichment and amplification methods used in viral metagenomics introduce unavoidable bias in viral diversity. This bias is due to shortcomings in existing viral enrichment methods and overshadowing by the more abundant viral populations. To reduce the complexity and improve the resolution of viral diversity, we developed a strategy coupling fluorescence-activated cell sorting (FACS) with random amplification and compared this to bulk metagenomics. This strategy was validated on both influent and effluent samples from a municipal wastewater treatment plant using the Modified Ludzack-Ettinger (MLE) process as the treatment method.We found that DNA and RNA communities generated using bulk samples were mostly different from those derived following FACS for both treatments before and after MLE. Before MLE treatment, FACS identified five viral families and 512 viral annotated contigs. Up to 43% of mapped reads were not detected in bulk samples. Nucleo-cytoplasmic large DNA viral families were enriched to a greater extent in the FACS-coupled subpopulations compared with bulk samples. FACS-coupled viromes captured a single-contig viral genome associated with Anabaena phage, which was not observed in bulk samples or in FACS-sorted samples after MLE. These short metagenomic reads, which were assembled into a high-quality draft genome of 46 kbp, were found to be highly dominant in one of the pre-MLE FACS annotated virome fractions (57.4%). Using bulk metagenomics, we identified that between Primary Settling Tank and Secondary Settling Tank viromes, Virgaviridae, Astroviridae, Parvoviridae, Picobirnaviridae, Nodaviridae, and Iridoviridae were susceptible to MLE treatment.

A panoramic view of the virosphere in three wastewater treatment plants by integrating viral‐like particle‐concentrated and traditional non‐concentrated metagenomic approaches

“…Focusing on small viruses, such as those in the Circoviridae and Parvoviridae families, typically under 30 nm in diameter, we found that the VPC approach was more effective in detecting Circoviridae populations than the NC approach (p < 0.001) (Figure S4B). This is consistent with the fact that many Circoviridae, small vertebrate-infecting viruses, are frequently detected in wastewater [21,22]. For Parvoviridae, however, there was no significant difference regarding the number of vOTUs detected between the two methods (p > 0.05) (Figure S4B).…”