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Review 3: "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 29, 2024
Review 3: "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
Description

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.

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Review: Targeted metagenomic sequencing can be effective in the detection of multiple human viruses in wastewater, but careful choice of nucleic acid extraction method should be made to enhance the efficacy of this method. Furthermore, while one extraction method may be optimal for detection of viruses using PCR-based methods, it may not be effective for detection by metagenomic methods.

This manuscript compares the impact of four wastewater nucleic acid extraction methods on the detection of human pathogenic viruses by hybrid-capture targeted metagenomic sequencing. The authors conclude that detection of human viruses is heavily influenced by the relative abundance of competing nucleic acid species in the sample, namely those of bacterial origin, which are more effectively depleted from the wastewater samples by the removal of solids after the release of viral particles. Overall, the data presented in the paper clearly support this conclusion, with a logical progression of analyses presented in a clear and concise manner to justify these claims. 

Authors present clear evidence that the Innovaprep ultrafiltration method, including wastewater solid removal, leads to the highest relative abundance of viral reads compared to bacterial reads, along with the highest coverage and richness of human virus. The Promega large-volume direct extraction, which also includes solids removal but without ultrafiltration to concentrate viral particles, leads to the second highest detection of human viruses and viral:bacterial ratio, further supporting the conclusion that solids removal effectively depletes completing bacterial nucleic acids. Authors also clearly demonstrate that the Innovaprep method results in the highest sensitivity for human virus detection despite having low absolute SARS-CoV-2 concentration, similar to or lower than other methods.

I propose one minor caveat regarding the conclusions about the low detection of SARS-CoV-2 in this study. In the final paragraph of section 3.6, authors mention the low coverage of SARS-CoV-2 in sequencing reads compared to other studies using the Illumina respiratory virus (RVOP) panel, which they propose is due to using the VSP hybrid-capture panel targeting a broader range of viruses in this study. However, it is possible that this is instead due to the higher prevalence of SARS-CoV-2 in the wastewater samples in other studies, which were carried out on samples from earlier in the SARS-CoV-2 pandemic, which is supported by the relatively low measured genome copies per μl of SARS-CoV-2 in all sample in this study. Authors also propose that the limited detection of SARS-CoV-2 relative to other enteric viruses is due to broad range of targets in the VSP panel reducing sensitivity of detection of SARS-CoV-2. It is more likely that this is due to the specific viruses targeted by the panel including enteric viruses which are in high abundance, leading to their dominance after hybrid capture, rather than the inherently broad nature of the panel’s targets. More careful wording could clarify this.

Comments
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