Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing

Abstract

Viral sewage metagenomics is a novel field of study used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency. In raw sewage human waste is mixed with household, industrial and drainage water, and virus particles are, therefore, only found in low concentrations. This necessitates a step of sample concentration to allow for sensitive virus detection. Additionally, viruses harbor a large diversity of both surface and genome structures, which makes universal viral genomic extraction difficult. Current studies have tackled these challenges in many different ways employing a wide range of viral concentration and extraction procedures. However, there is limited knowledge of the efficacy and inherent biases associated with these methods in respect to viral sewage metagenomics, hampering the development of this field. By the use of next generation sequencing this study aimed to evaluate the efficiency of four commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS® miniMAG®, or PowerViral® Environmental RNA/DNA Isolation Kit) to determine the viriome in a sewage sample. We found a significant influence of concentration and extraction protocols on the detected viriome. The viral richness was largest in samples extracted with QIAamp Viral RNA Mini Kit or PowerViral® Environmental RNA/DNA Isolation Kit. Highest viral specificity were found in samples concentrated by precipitation with polyethylene glycol or extracted with Nucleospin RNA XS. Detection of viral pathogens depended on the method used. These results contribute to the understanding of method associated biases, within the field of viral sewage metagenomics, making evaluation of the current literature easier and helping with the design of future studies.

Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 700 sequencing libraries

“…Many of these viruses are non-human and identification of these would not be expected in human cancer tissue samples. Some of the viral sequences have previously been suggested to be contamination, such as Pepino mosaic virus [33], Gallid herpes virus, Pandoravirus, Citrobacter phage [34] and Rotavirus [32,35,36]. More viral sequences significantly associated with laboratory components were identified among reads than contigs, a probable explanation being the low depth of coverage of genomes, making assembly of viral reads into contigs infrequent.…”

Membrane Bioreactor-Based Wastewater Treatment Plant in Saudi Arabia: Reduction of Viral Diversity, Load, and Infectious Capacity

“…However, similar to other concentration methods, e.g., cesium chlorine ultracentrifugation, full recovery of particles in wastewater cannot be achieved by TFF. Studies have shown that different methods of viral purification result in different families being detected [24]. TFF has been chosen for this current study for its relatively high recovery rate and is common in studies involving wastewater, hence allowing for direct comparison between studies [25,26].…”

“…While more studies have to be carried out to determine if their pathogenicity persisted post treatment, 6 of those families (i.e., Adenoviridae, Picornaviridae, Parvovoridae, Astroviridae, Picornabviridae, and Polyomaviridae) have also been reported as potentially pathogenic viral families in raw sewage by another study [24]. However, the disparity in the number of pathogenic viruses detected could be due to the difference in geographic location, genetic-make up, diet, population sizes, and purification methods [24]. Furthermore, more viral families were noted in the Influent-Wash as compared to the Influent-Retentate (Figure 2).…”

Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples

“…Numerous protocols for viral enrichment and genome amplification have been described in literature [5][6][7]. Commonly employed protocols such as sample filtration [8], nuclease digestion, ultracentrifugation, and random pre-amplification of RNA or DNA in separate reactions would be particularly useful for increasing the signal-to-noise ratio in the viral analysis of biological samples in which the levels of nucleic acid background are high.…”

“…Amplicon-based NGS only detected pre-defined targets, thus possibly missing viruses or novel virus strains. Apart from these methods, a crucial step in the molecular detection of viruses in clinical specimens is the efficient extraction of viral nucleic acids [7]. Higher virus-related yields of the extracts meant better sensitivity in the subsequent detection analysis.…”