Targeted amplification and genetic sequencing of the severe acute respiratory syndrome coronavirus 2 surface glycoprotein

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon dropout. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitors primer conservation as SARS-CoV-2 evolves. Despite being designed during the beta wave, the selected primers remain >99% conserved through Omicron as of 19 October 2023. To validate the final design, we compared targeted S-gene data to National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. This method can serve as a complement to whole-genome surveillance or can be leveraged where only S-gene sequencing is of interest. IMPORTANCE The COVID-19 pandemic was accompanied by an unprecedented surveillance effort. The resulting data were and will continue to be critical for surveillance and control of SARS-CoV-2. However, some genomic surveillance methods experienced challenges as the virus evolved, resulting in incomplete and poor quality data. Complete and quality coverage, especially of the S-gene, is important for supporting the selection of vaccine candidates. As such, we developed a robust method to target the S-gene for amplification and sequencing. By focusing on the S-gene and imposing strict coverage and quality metrics, we hope to increase the quality of surveillance data for this continually evolving gene. Our technique is currently being deployed globally to partner laboratories, and public health representatives from 79 countries have received hands-on training and support. Expanding access to quality surveillance methods will undoubtedly lead to earlier detection of novel variants and better inform vaccine strain selection.


Major Comments:
1.There is quite a bit of data generated and work put into this project.However, all the data in the 'Results' section are in the Supplemental Material.This makes it very difficult for the readers to grasp the material and truly appreciate the work and findings.Please consider moving some of the supplemental data into the main text.I think some of the key figures from the supplemental data include the conservation of your primers (lines 125-133), the LoD, Method Validation (line 149), and Phylogenetics (line 212).Rather than putting all the data, please find representative data per each section of the 'Results' section to make different figures.Then, any extra data can be referred to in Supplemental Material.
2. Certain areas of the 'Results' can be further elaborated and described.For example, lines 138-140 where it simply states "We performed 14 Nanopore sequencing runs to validate and characterize our method.A summary of these runs is available in the supplemental materials" is not sufficient for the 'Results' section paragraph and should really be elaborated given that these runs are important for the paper.
3. The abstract states, "While it is adaptable to other sequencing platforms, the Nanopore platform validated here...".I would suggest making a separate figure and section in the 'Results' that compares performance of different platforms.If not, then this statement in the 'Abstract' is an overstatement and this manuscript should mainly be focused on Oxford Nanopore technologies, which has merit in itself as well.Please revise accordingly.4. Much of the 'Discussion' was written as a description for 'ongoing projects' and 'future steps' with this project, which is acceptable.However, some of the key components for a typical 'discussion' section appears to be missing.For example, how does your protocol compare to other assays ran on Oxford Nanopore?What are the limitations of this study?
Minor comments: 1.Some of the supplemental data has the Qiagen header in the data and it appears like these are raw printouts of the data.Is this allowed for publication since Qiagen's logo and trademark is being used?Please present the data in another way and make your own figures with the raw data.

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Summary of Key Findings (200-250 words)
Thank you for the opportunity to review this manuscript.In this study, Keller et al. describe the challenges associated with sequencing the SARS-CoV-2 S-gene in the context of mutations across the COVID-19 pandemic, and describe the development and validation of a targeted method to amplify and sequence the S-gene.Furthermore, the authors explain the potential public health impact of this method due to the fact that the total number of primers used is four, and the number of primers within the coding region of the S-gene is two.This is a well-written manuscript that contributes to the growing body of COVID-19 evidence, and has an important focus on accessibility, surveillance, and global public health.

Major Concerns (at most 5-6): None
Minor Concerns (at most 5-20 in bullet points): 1. Line 175-176: If possible, it would be great if a citation could be added to the end of this sentence to elucidate/support the minimum amount of mutations needed to be considered acceptable for surveillance (or something that references public health sequencing recommendations).2. Next generation sequencing is abbreviated throughout the manuscript.Consider doing the same with whole-genome sequencing, if appropriate.

Editor
This is a strong project nonetheless but perhaps, could be presented in a better way.The group needs to transfer some supplemental data into figures for the main text and in some cases, elaborate and describe the data a bit more and not assume the audience can understand the data.
We thank the editor for the comments and for refereeing this review.We too believe this is a strong project.To clarify the work done, we have incorporated much of the suggested feedback.Primarily, we have moved some figured from the supplemental into the main text and have expanded the results sections discussing those key finding.We have also expanded the discussion to include limitations.We disagree with reviewer #2 regarding the inclusion of LOD data for a third sequencing method.We have elaborated in the response to that comment, but to briefly summarize, we feel that experiment would not reveal any practical information as the LOD is primarily a property of the RT-PCR and the influence of a different sequencer has less influence than how many samples are indexed for a single run.
Reviewer #1 (Comments for the Author): Thank you for the opportunity to review this manuscript.In this study, Keller et al. describe the challenges associated with sequencing the SARS-CoV-2 S-gene in the context of mutations across the COVID-19 pandemic, and describe the development and validation of a targeted method to amplify and sequence the S-gene.Furthermore, the authors explain the potential public health impact of this method due to the fact that the total number of primers used is four, and the number of primers within the coding region of the S-gene is two.This is a well-written manuscript that contributes to the growing body of COVID-19 evidence, and has an important focus on accessibility, surveillance, and global public health.
Major Concerns: None Minor Concerns: 1.Line 175-176: If possible, it would be great if a citation could be added to the end of this sentence to elucidate/support the minimum amount of mutations needed to be considered acceptable for surveillance (or something that references public health sequencing recommendations).
As suggested, we've added a reference to Genomic sequencing of SARS-CoV-2: A guide to implementation for maximum impact on public health.
2.Next generation sequencing is abbreviated throughout the manuscript.Consider doing the same with whole-genome sequencing, if appropriate.We thank the reviewer for the suggestion.Whole-genome sequencing has been abbreviated to WGS throughout the manuscript Reviewer #2 (Comments for the Author): Authors have described a robust method to target the S-gene for amplification and sequencing of SARS-CoV-2.The methods appear useful in making decisions on virus mutations and evolutions of essential regions such as the spike protein.The manuscript generally appears sound with good technical and scientific details.Authors have indicated progress toward making these methods available to LMICs.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred