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

Abstract

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.

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

“…In support of this observation, the ratio of target-to-nontarget in the input nucleic acids has previously been shown to affect the success of probe-capture sequencing . Accordingly, measurement of this ratio (via dPCR and/or Qubit) before library preparation and enrichment might be a useful predictor for the success of sequencing . Additional parameters of interest include target virus concentration, nucleic acid integrity, fragment size distribution, and nontarget sequence composition.…”

“…However, several studies using the narrower RVOP panel found remarkably high coverages of SARS-CoV-2, surpassing other respiratory human viruses included in the panel. ,, Another study also found that the share of sequences derived from SARS-CoV-2 was higher when using the Respiratory Virus Oligo Panel (RVOP) than the broader Respiratory Pathogen ID/AMR Panel (RPIP), which included other high-abundance targets . Although this high sequencing recovery of SARS-CoV-2 may have been partially due to the higher prevalence at the sampling time, future work is needed to design and benchmark custom probe panels that balance target diversity and sequencing sensitivity for early detection of emerging virus strains …”

“… 59 Accordingly, measurement of this ratio (via dPCR and/or Qubit) before library preparation and enrichment might be a useful predictor for the success of sequencing. 61 Additional parameters of interest include target virus concentration, nucleic acid integrity, fragment size distribution, and nontarget sequence composition. Future studies should statistically compare these evaluation metrics with the final sequencing performance on a larger number of samples.…”

“… 60 Although this high sequencing recovery of SARS-CoV-2 may have been partially due to the higher prevalence at the sampling time, future work is needed to design and benchmark custom probe panels that balance target diversity and sequencing sensitivity for early detection of emerging virus strains. 61 …”