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Review 2: "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 2: "Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater"
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key-enterThis Pub is a Review of
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: This study compares the impact of four different virus concentration/extraction methods that are applied to sequencing of DNA and RNA in wastewater samples. The authors have carefully considered the selection of methods based on those that are currently used in wastewater surveillance. In an ideal study, perhaps a more exhaustive selection of methods could have been implemented, but this doesn’t take away from the significance of the comparison between the methods tested. There is some limitation due to only using the Allprep Power Viral DNA/RNA kit, since the selection of this kit could impact results. However, keeping this constant while varying the upstream methods is logical. Perhaps a future study could look at the impact of the extraction kit. It is also worth noting that the number of wastewater samples is relatively low at just three. While this is fine for a comparative study such as the one shown, testing across a wider range of wastewater samples would improve the strength of the evidence. As noted by the authors, the composition of each sample can change considerably, in terms of the abundance of particular viruses. Therefore a long-term monitoring study could further substantiate the conclusion that was reached with regards to the most effective method. Overall, this is a good quality study with a high level of relevance to wastewater-based surveillance.

In summary, this manuscript is relevant to wastewater-based surveillance of human viruses. Sequencing of these viruses is valuable for understanding the emergence of different variants of concern. This study contributes to improving the quality of sequencing data from wastewater samples.

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