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Review 1: "Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater"

The reviewers found this study to be well designed and conducted, with reliable results relevant to researchers working in the field of wastewater surveillance. Very minor comments were made regarding the choice of the four methods.

Published onFeb 27, 2024
Review 1: "Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater"
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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

Abstract 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.Synopsis Four concentration/extraction methods combined with probe-capture sequencing of human viruses in raw wastewater were compared. Innovaprep ultrafiltration with solids removal had the best performance for human virus detection sensitivity, richness, and recovery of near-complete genomes.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.


Review: Monitoring for human viruses in wastewater presents an effective strategy for tracking the emergence of pathogenic viruses. Sequencing viruses in wastewater provides more useful information than the PCR-based detection methods. However, the complexity and heterogeneity of wastewater, the presence of a plethora of microorganisms and bacterial viruses, and very low concentrations of human viruses pose significant challenges for the selective concentration of human viruses from wastewater for nucleic acid extraction and sequencing. As unbiased sequencing of the isolated nucleic acid does not efficiently capture the human viruses, target-enrichment methods are gaining traction. Hence, the development of a robust, universally applicable, end-to-end protocol for virus extraction, concentration, and sequencing from wastewater is a major challenge. In this context, this manuscript assesses the utility of four virus extraction and concentration methods, namely, Innovaprep, Nanotrap, Promega, and Solids extraction for targeted sequencing. The study concludes that while the Innovaprep method offers a better capture of most viruses, Promega and Nanodrop methods fared better for detecting SARS-CoV-2 by the dPCR method.

The usage of the same sample volume for four extraction methods made the comparisons straightforward. The viruses that are shed in high concentrations were detected by all methods.

The Innovaprep samples yielded enough reads to obtain near-complete assemblies of 7 genomes with JC polyomavirus phylogeny and strain variation yielding potentially epidemiological information. A comparison with dPCR further shows that the Promega method shows a better recovery of viral RNA than IP and also no correlation was observed between the target concentrations by the dPCR method with sequence read counts, which is expected. While the removal of solids from wastewater significantly improves the recovery of human viruses by sequencing, it reduces the sensitivity of dPCR-based detection. The study also concludes that there is much scope for bettering the probe panel. Because many human viruses, and endemic and rare, a universal probe panel with a limited number of viruses may not be suitable for wastewater-based surveillance in all parts of the world.

This is a well-done study and useful for researchers working in this area. The protocols and modifications (including bioinformatics methods) have been articulated adequately. The manuscript is written well. A major takeaway from this study is that researchers have to carefully choose the method based on the endpoint assay and overall goal. Some suggestions are as follows:

  • Lines 103-105: For the study, authors have used the commercially available Bovine coronavirus (BCoV) vaccine powder from MERCK as a standard for spiking the samples. Standardized NGS mock viral communities with varying surface characteristics such as the ones available with ATCC would have been more informative on the recovery efficiencies of individual viruses by different methods. 

  • Section 2.2: Lines 106-112: It is not clear why these four methods were selected for virus concentration and extraction. Some discussion on this in the first part of the results and discussion section (lines 243-251) would be helpful to the uninitiated as several new methods have been reported post-COVID.

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