Evaluation of methods for the concentration and extraction of viruses from sewage in the context of metagenomic sequencing

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

Evaluation of methods for the concentration and extraction of viruses from sewage in the context of metagenomic sequencing

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

Detection of pathogenic viral families.

<p>Heatmap of the relative abundance of 14 human pathogenic viral families, detected by the 16 different concentration/extraction combinations. The numbers within each cell represents reads per million. The colours range from green = no detection, to red = high relative abundance.</p

The influence of extraction method on the viral community composition.

<p>PCA plots made by using the relative abundances of the nine most abundant viral families. Separate PCAs were done for (A) samples concentrated with MAF, (B) SMF, (C) GW, and (D) PEG. Sample replicates were individually plotted and grouped according to the extraction method. In cases where only two samples were present, no ellipse representing the cluster was drawn.</p

Viral species richness.

<p>(A) Viral Chao 1 species richness of the tested concentration methods, and (B) extraction methods. Each boxplot was made from 12 individual samples (including the four extraction/concentration methods with three replicates each). The bar, box, whiskers and circles represents median, inter-quartile range, inter-quartile range times 1.5, and outliers, respectively. Asterisks represent significance level of a pairwise t-test with “Holm-Bonferroni” adjusted p-values. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.</p