Effectiveness of passive sampling for the detection and genetic characterization of human viruses in wastewater

Mejías-Molina, Cristina; Pico-Tomàs, Anna; Beltran-Rubinat, Andrea; Martínez-Puchol, Sandra; Corominas, Lluís; Rusiñol, Marta; Bofill-Mas, Sı́lvia

doi:10.1039/d2ew00867j

Cited by 16 publications

(11 citation statements)

References 56 publications

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“…All human virus species detected (>10 reads per species) in at least one sample were compared across the four methods (Figure ). Some viruses were consistently detected by all methods, including human polyomavirus, mastadenovirus, mamastrovirus 1, and norwalk virus, which were included in the VSP probe set and are known to be shed at high concentrations in human waste. ,,,,,,− RNA virus species, including severe acute respiratory syndrome-related coronavirus, sapporo virus, and enteroviruses, were not detected in NT and Solids samples. Different trends were also observed among virus species within the same genus.…”

Section: Resultsmentioning

confidence: 97%

“…JC polyomavirus composite genomes were assembled in samples from three concentration/extraction methods (IP, PMG, and NT) and multiple replicates (Figure S5b). The recovery of JC polyomavirus genomes is perhaps unsurprising given that approximately 40% of the population sheds the virus through urine . Also, as a nonenveloped DNA virus with a circular genome, JC polyomavirus is highly resistant to environmental stress and exonuclease activity .…”

Section: Resultsmentioning

confidence: 99%

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Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Jiang,

Wang,

et al. 2024

Environ. Sci. Technol.

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Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/ extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Jiang,

Wang,

et al. 2024

Environ. Sci. Technol.

View full text Add to dashboard Cite

show abstract

“…All human virus species detected (>10 reads per species) in at least one sample were compared across the four methods (Figure 4). Some viruses were consistently detected by all methods, including human polyomavirus, mastadenovirus, mamastrovirus 1, and norwalk virus, which are known to be shed at high concentrations in human waste 5, 9, 10, 13, 23, 48, 52–55 . RNA virus species, including severe acute respiratory syndrome-related coronavirus, sapporo virus, and enteroviruses were not detected in NT and Solids samples.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of the impact of concentration and extraction methods on the targeted sequencing of human viruses from wastewater

Jiang,

Wang,

et al. 2024

Preprint

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Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of most near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by dPCR compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased by using methods that reduce non-target nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

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“…Child et al applied both RVOP and RPIP in parallel and reported lower coverage of SARS-CoV-2 with RPIP, likely due to competition for sequencing with other targets . Several other studies applied very broad panels covering major groups of human viruses and reported that the predominant human virus was Mamastrovirus followed by other enteric viruses, while respiratory viruses were at lower relative abundance sometimes by orders of magnitude. ,− , Although differences in samples, sample processing, and bioinformatics make direct comparisons impossible (Table ), these results point to trade-offs in probe panel selection.…”

Section: Probe Set Selection Hybridization and Sequencing Depthmentioning

confidence: 99%

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

Kantor,

Jiang

2024

Environ. Sci. Technol.

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Until recently, wastewater-based monitoring for pathogens of public health concern primarily used PCR-based quantification methods and targeted sequencing for specific pathogens (e.g., SARS-CoV-2). In the past three years, researchers have expanded sequencing to monitor a broad range of pathogens, applying probe capture enrichment to wastewater. The goals of those studies included (1) monitoring and expanding fundamental knowledge of disease dynamics for known pathogens and (2) evaluating the potential for early detection of emerging diseases resulting from zoonotic spillover or novel viral variants. Several studies using off-the-shelf probe panels designed for clinical and environmental surveillance reported that enrichment increased virus relative abundance but did not recover complete genomes for most nonenteric viruses. Based on our experience and recent results reported by others using these panels for wastewater, clinical, and synthetic samples, we discuss challenges and technical factors that affect the rates of false positive and false negative results. We identify trade-offs and opportunities throughout the workflow, including in wastewater sample processing, probe panel design, and bioinformatic analysis. We suggest tailored methods of virus concentration and background removal, carefully designed probe panels, and multithresholded bioinformatics analysis.

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Effectiveness of passive sampling for the detection and genetic characterization of human viruses in wastewater

Abstract: Wastewater-based epidemiology is a promising tool to anticipate potential viral outbreaks not only at city-scale but also at precincts-scale or even buildings.

Cited by 16 publications

References 56 publications

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Evaluation of the impact of concentration and extraction methods on the targeted sequencing of human viruses from wastewater

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

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