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

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.

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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.