Gene Sequencing: Key for Analyzing Mutation in SARS-CoV-2
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Genomic sequencing is a sole platform that recognizes and characterizes novel mutants of microorganisms existing in various clinical samples. The main objective of this article is to summarize and provide relevant information regarding genomic sequencing platforms that are widely applied for analyzing mutations in SARS-CoV-2. Various data and literature were reviewed from scientific database like PubMed, Scopus, Google scholars and internet in order to provide collective and reliable information on genomic sequencing. Various Variants of Concern (VOC) and Variants of Interest (VOI) with higher transmissibility and infectivity are detected around the globe among which Nepal is also at high risk stage. According to the Ministry of Health and Population (MOHP) delta variants (B.1.617.2), alpha variants (B.1. 1.7), kappa variants(B.1.617.1) and newly emerged sub lineage of delta variants (A.Y.1) are the predominant one that are circulating in the country. Therefore, severe acute respiratory syndrome coronavirus 2 whole genome sequencing, Next generation sequencing (Illumina MiSeq. and Oxford Nanopore Minion) or Sanger method based on amplicon sequencing are the best optional platforms for the detection of VOC and VOI and to continue monitoring of widespread SARS-CoV-2 for proper therapy and prevention of COVID-19. Besides, variations occurring in the fragment of virus genome may produce gene dropout obstructing with laboratory molecular assays and epidemiological surveys, therefore it is very crucial to detect those markers with the aid of gene sequencing methods in order to establish higher molecular testing strategies.
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Please include more rigour criteria and resources in your methods section, as highlighted by the SciScore reports. Including RRIDs and negative statements to explain why things were not performed should increase the rigour and reproducibility of your work. You can find tips on how to improve your article here: https://sciscore.com/reports/Core-Report.php Please provide more detail in the Methods section and ensure that software is consistently cited and its version and parameters included. The reviewers raise concerns regarding the scientific rigour and experimental design of the work. The paper is poorly structured and written, which has prevented a proper assessment of the research done.
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Comments to Author
1. Literature analysis or discussion * Various VOCs are included in Table 1, however, this does not include any Omicron variants which have been circulating since December 2021 and made up a large proportion of VOCs in 2022. Therefore, Omicron VOCs should be included in this table, especially as the literature search included papers from 2022. Also studies have found reduced enrichment efficiency of PCR-tiling amplicon protocols with Midnight and Artic primers due to large number of mutations on S gene which is important to include in the impact on overall performance of molecular diagnostics column. * Section 1: Sequencing. This section describes the different sequencing techniques. This does not give examples of how studies have used these techniques to sequence for SARS-CoV-2 and if these studies …
Comments to Author
1. Literature analysis or discussion * Various VOCs are included in Table 1, however, this does not include any Omicron variants which have been circulating since December 2021 and made up a large proportion of VOCs in 2022. Therefore, Omicron VOCs should be included in this table, especially as the literature search included papers from 2022. Also studies have found reduced enrichment efficiency of PCR-tiling amplicon protocols with Midnight and Artic primers due to large number of mutations on S gene which is important to include in the impact on overall performance of molecular diagnostics column. * Section 1: Sequencing. This section describes the different sequencing techniques. This does not give examples of how studies have used these techniques to sequence for SARS-CoV-2 and if these studies have been successful or if there were any limitations. Are there differences between the studies e.g. sample type, method used e.g. library preparation, sequencing technologies e.g. MiSeq, NovaSeq, bioinformatics needed. Are there benefits of each method over the other methods? Do the authors have recommendations over which sequencing technique should be used for different variants? This information could be nicely summarized in a table. 2. Any other relevant comments 1. Abstract * The abstract should be rewritten, currently it is hard to follow. * Line 19 - 20: The minimal set of D80A, D215G, E484K, N501Y, and A701V: what are these a set of? This should be explained. 2. Introduction * Line 31 - 34: Paper states that it is crucial to analyse different VOCs, however, this should be expanded to explain why this is important. * Line 37 - 38: The whole genome sequencing (WGS) method is insufficient for determining the frequency of VOCs in the community and for quickly identifying mutants for health promotion solutions [2]. - This is not the correct reference for this statement, this is about increased transmissibility of SARS-CoV-2 lineage B.1.1.7 rather than determining the frequency of VOCs * Line 39 - 40: WGS still a somewhat expensive assay with a significant decline in the value - This statement is contradictory. Is it expensive or has the price declined making it more affordable? * Line 42: References 3 and 4 are not correct for this statement * Line 46: Reference 6 downloaded all the sequences from NCBI for analysis - they did not perform a PCR based protocol before sequencing the samples and so this reference is not correct for this statement. * Line 47: Various groups have previously invented or are presently inventing and assessing a foresaid methods - what are these methods that the groups have invented/inventing? This could be discussed further with references. * A sentence should be included to state what the aim/objective of the review. 3. Methods * Searches were made across different databases for papers. Were the available papers screened and criteria applied for the inclusion or exclusion of papers? 4. Results * Table 1 needs reformatted, some words are joined together, especially in the mutations and impact on overall performance of molecular diagnostics columns. * The conclusion for Beta and Gamma in the impact on overall performance of molecular diagnostics column in Table 1 was minimal effect but then states no records on the effect of mutations on the overall assay and can affect assays that concentrate on S gene sequences - how can you conclude then that there is minimal effect? This table is also only based on two references, are there other studies that report on the overall assay? * Figure 1 is not from reference 20, what is the correct reference for this figure? * Line 102: Vogel et al cited in text but reference provided was Munoz et al. Vogel et al was not included in the bibliography. * Line 176: Reference 24 is incorrect for this sentence, it is referring to long read sequencing rather than hybrid enrichment-capture sequence analysis. * Section 1.7: Fourth-generation sequencing - Oxford nanopore technology is considered third generation/long read sequencing (reference 24). * Line 204: Reference 28 is not correct for this sentence. * Section 2.1 S gene rejection or targeting exclusion - Reference 29 details a N gene dropout for B 1.1.7 lineage not S gene dropout. The information on line 207 - 211 is incorrect. 5. Conclusion * This is an overview on why it is important to sequence the SARS-CoV-2 VOCs, this information could be condensed. * There is no conclusion regarding the different sequencing technologies/methods and the success of these for sequencing SARS-CoV-2. 6. References * References 5, 7, 13, 18, 19, 23, 29 are preprints; this has not been included in the bibliography. Some of these have been published so the published reference and not the preprint should be cited.
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: No animal work involved in this study
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Please rate the quality of the presentation and structure of the manuscript
Very poor
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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