Looking into sewage: how far can metagenomics help to detect human enteric viruses?

Abstract

The impact of human sewage on environmental and food contamination constitutes an important safety issue. Indeed, human sewage reflects the microbiome of the local population, and a variety of human viruses can be detected in wastewater samples. Being able to describe the diversity of viruses present in sewage will provide information on the health of the surrounding population health and will help to prevent further transmission. Metagenomic developments, allowing the description of all the different genomes present in a sample, are very promising tools for virome analysis. However, looking for human enteric viruses with short RNA genomes which are present at low concentrations is challenging. In this study we demonstrate the benefits of performing technical replicates to improve viral identification by increasing contig length, and the set-up of quality criteria to increase confidence in results. Our approach was able to effectively identify some virus sequences and successfully describe the viral diversity. The method yielded full genomes either for norovirus, enterovirus and rotavirus, even if, for these segmented genomes, combining genes remain a difficult issue. Developing reliable viromic methods is important as wastewater sample analysis provides an important tool to prevent further virus transmission by raising alerts in case of viral outbreaks or emergence.

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

“…For example, more complex sample types of public health relevance remain untested, such as stool, wastewater, or shellfish. Promising results have been found from ultra-deep sequencing of wastewater enriched with a broad viral discovery probe panel, yielding three partial HAV genomes (1,482–6,729 nts) ( 58 , 59 ). Cost may also be a limiting factor for wider implementation.…”

Considerations and Opportunities for Probe Capture Enrichment Sequencing of Emerging Viruses from Wastewater

“…To determine that a virus present in wastewater is novel, de novo assembly of at least a partial viral genome would likely be required. However, given that assembled contig length is related to sequencing coverage, assembly is likely to be infeasible for an emerging virus at low coverage. Instead, read-based classification might be used to alert users to an unusual spike in viruses classified at a higher taxonomic level (e.g., Beta-coronaviruses) .…”

“…To predict sequencing success after concentration and extraction, the target-to-background mass ratio should be taken into account, not just the target concentration ( Figure 1 c). When target abundance is low, sequencing replicates can improve sensitivity, 39 but replication is obviously limited by cost. To control for method failure, PCR-based quantification of endogenous fecal viral indicators (e.g., pepper mild mottle virus or crAssphage, now Carjivirus) could be used to confirm successful concentration and extraction.…”