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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is adaptively evolving to ensure its persistence within human hosts. It is therefore necessary to continuously monitor the emergence and prevalence of novel variants that arise. Importantly, some mutations have been associated with both molecular diagnostic failures and reduced or abrogated next-generation sequencing (NGS) read coverage in some genomic regions. Such impacts are particularly problematic when they occur in genomic regions such as those that encode the spike (S) protein, which are crucial for identifying and tracking the prevalence and dissemination dynamics of concerning viral variants. Targeted Sanger sequencing presents a fast and cost-effective means to accurately extend the coverage of whole-genome sequences. We designed a custom set of primers to amplify a 401 bp segment of the receptor-binding domain (RBD) (between positions 22698 and 23098 relative to the Wuhan-Hu-1 reference). We then designed a Sanger sequencing wet-laboratory protocol. We applied the primer set and wet-laboratory protocol to sequence 222 samples that were missing positions with key mutations K417N, E484K, and N501Y due to poor coverage after NGS sequencing. Finally, we developed SeqPatcher, a Python-based computational tool to analyse the trace files yielded by Sanger sequencing to generate consensus sequences, or take preanalysed consensus sequences in format, and merge them with their corresponding whole-genome assemblies. We successfully sequenced 153 samples of 222 (69 %) using Sanger sequencing and confirmed the occurrence of key beta variant mutations (K417N, E484K, N501Y) in the S genes of 142 of 153 (93 %) samples. Additionally, one sample had the Y508F mutation and four samples the S477N. Samples with RT-PCR scores ranging from 13.85 to 37.47 (mean=25.70) could be Sanger sequenced efficiently. These results show that our method and pipeline can be used to improve the quality of whole-genome assemblies produced using NGS and can be used with any pairs of the most used NGS and Sanger sequencing platforms.

Funding
This study was supported by the:
  • National Human Genome Research Institute (Award U24HG006941)
    • Principle Award Recipient: Tuliode Oliveira
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2022-03-16
2024-06-18
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References

  1. Li Q, Wu J, Nie J, Zhang L, Hao H et al. The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity. Cell 2020; 182:1284–1294 [View Article] [PubMed]
    [Google Scholar]
  2. Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J et al. Tracking changes in SARS-CoV-2 spike: evidence that d614g increases infectivity of the COVID-19 virus. Cell 2020; 182:812–827 [View Article] [PubMed]
    [Google Scholar]
  3. Zhang L, Jackson CB, Mou H, Ojha A, Peng H et al. SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity. Nat Commun 2020; 11:6013 [View Article] [PubMed]
    [Google Scholar]
  4. He Y, Zhou Y, Liu S, Kou Z, Li W et al. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine. Biochem Biophys Res Commun 2004; 324:773–781 [View Article] [PubMed]
    [Google Scholar]
  5. VanBlargan LA, Goo L, Pierson TC. Deconstructing the antiviral neutralizing-antibody response: implications for vaccine development and immunity. Microbiol Mol Biol Rev 2016; 80:989–1010 [View Article] [PubMed]
    [Google Scholar]
  6. Yin C. Genotyping coronavirus SARS-CoV-2: methods and implications. Genomics 2020; 112:3588–3596 [View Article] [PubMed]
    [Google Scholar]
  7. Tang X, Wu C, Li X, Song Y, Yao X et al. On the origin and continuing evolution of SARS-CoV-2. Natl Sci Rev 2020; 7:1012–1023 [View Article] [PubMed]
    [Google Scholar]
  8. Cotten M, Lule Bugembe D, Kaleebu P, V T Phan M. Alternate primers for whole-genome SARS-CoV-2 sequencing. Virus Evol 2021; 7:veab006 [View Article] [PubMed]
    [Google Scholar]
  9. Illumina Key differences between next-generation sequencing and Sanger sequencing: Understanding when NGS can be a more effective option; 2021 https://emea.illumina.com/science/technology/next-generation-sequencing/ngs-vs-sanger-sequencing.html
  10. Nasir JA, Kozak RA, Aftanas P, Raphenya AR, Smith KM et al. A comparison of whole genome sequencing of SARS-CoV-2 using amplicon-based sequencing, random hexamers, and bait capture. Viruses 2020; 12:E895 [View Article] [PubMed]
    [Google Scholar]
  11. Hagemann IS. Overview of technical aspects and chemistries of next-generation sequencing. In Clinical Genomics Elsevier; 2015 pp 3–19
    [Google Scholar]
  12. Borges V, Sousa C, Menezes L, Gonçalves AM, Picão M et al. Tracking SARS-CoV-2 lineage B.1.1.7 dissemination: insights from nationwide spike gene target failure (SGTF) and spike gene late detection (SGTL) data, Portugal, week 49 2020 to week 3 2021. Euro Surveill 2021; 26: [View Article] [PubMed]
    [Google Scholar]
  13. Bal A, Destras G, Gaymard A, Stefic K, Marlet J et al. Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69–V70, France, August to December 2020. Euro Surveill 2021; 26: [View Article]
    [Google Scholar]
  14. Sapoval N, Mahmoud M, Jochum MD, Liu Y, Elworth RAL et al. SARS-CoV-2 genomic diversity and the implications for qRT-PCR diagnostics and transmission. Genome Res 2021; 31:635–644 [View Article] [PubMed]
    [Google Scholar]
  15. Nadalin F, Vezzi F, Policriti A. GapFiller: a de novo assembly approach to fill the gap within paired reads. BMC Bioinformatics 2012; 13 Suppl 14:S8 [View Article] [PubMed]
    [Google Scholar]
  16. Greaney AJ, Loes AN, Crawford KH, Starr TN, Malone KD et al. Comprehensive mapping of mutations in the sars-cov-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies. Cell Host & Microbe 2021
    [Google Scholar]
  17. Wang Z, Schmidt F, Weisblum Y, Muecksch F, Barnes CO et al. mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. Nature 2021; 592:616–622 [View Article]
    [Google Scholar]
  18. Cheng MH, Krieger JM, Kaynak B, Arditi M, Bahar I. Impact of south african 501.v2 variant on SARS-CoV-2 spike infectivity and neutralization: a structure-based computational assessment. BioRxiv 2021
    [Google Scholar]
  19. Koressaar T, Remm M. Enhancements and modifications of primer design program Primer3. Bioinformatics 2007; 23:1289–1291 [View Article] [PubMed]
    [Google Scholar]
  20. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC et al. Primer3--new capabilities and interfaces. Nucleic Acids Res 2012; 40:e115 [View Article] [PubMed]
    [Google Scholar]
  21. Kõressaar T, Lepamets M, Kaplinski L, Raime K, Andreson R et al. Primer3_masker: integrating masking of template sequence with primer design software. Bioinformatics 2018; 34:1937–1938 [View Article] [PubMed]
    [Google Scholar]
  22. Brzoska PM, Brown C, Cassel M, Ceccardi T, Di Francisco V et al. An efficient and high-throughput approach for experimental validation of novel human gene predictions. Genomics 2006; 87:437–445 [View Article] [PubMed]
    [Google Scholar]
  23. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 2004; 5:113 [View Article] [PubMed]
    [Google Scholar]
  24. Kent WJ. BLAT--the BLAST-like alignment tool. Genome Res 2002; 12:656–664 [View Article] [PubMed]
    [Google Scholar]
  25. World Health Organization Genomic sequencing of SARS-CoV-2: a guide to implementation for maximum impact on public health Geneva: World Health Organization; 2021
    [Google Scholar]
  26. Ewels PA, Peltzer A, Fillinger S, Patel H, Alneberg J et al. The nf-core framework for community-curated bioinformatics pipelines. Nat Biotechnol 2020; 38:276–278 [View Article] [PubMed]
    [Google Scholar]
  27. Cock PJA, Antao T, Chang JT, Chapman BA, Cox CJ et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 2009; 25:1422–1423 [View Article] [PubMed]
    [Google Scholar]
  28. Nurk S, Meleshko D, Korobeynikov A, Pevzner PA. metaSPAdes: a new versatile metagenomic assembler. Genome Res 2017; 27:824–834 [View Article] [PubMed]
    [Google Scholar]
  29. Li D, Liu C-M, Luo R, Sadakane K, Lam T-W. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 2015; 31:1674–1676 [View Article] [PubMed]
    [Google Scholar]
  30. Cleemput S, Dumon W, Fonseca V, Abdool Karim W, Giovanetti M et al. Genome Detective Coronavirus Typing Tool for rapid identification and characterization of novel coronavirus genomes. Bioinformatics 2020; 36:3552–3555 [View Article] [PubMed]
    [Google Scholar]
  31. Deforche K. An alignment method for nucleic acid sequences against annotated genomes. BioRxiv 2017
    [Google Scholar]
  32. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 2018; 34:3094–3100 [View Article] [PubMed]
    [Google Scholar]
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