Walker, A., Houwaart, T., Wienemann, T., Vasconcelos, M.K., Strelow, D., Senff, T., Hülse, L., Adams, O., Andree, M., Hauka, S., Feldt, T., Jensen, B.E., Keitel, V., Kindgen-Milles, D., Timm, J., Pfeffer, K. and Dilthey, A.T. (2020) Genetic Structure of SARS-CoV-2 Reflects Clonal Superspreading and Multiple Independent Introduction Events, North-Rhine Westphalia, Germany, February and March 2020. European Communicable Disease Bulletin, 25, Article ID: 2000746.
https://doi.org/10.2807/1560-7917.ES.2020.25.22.2000746

has been cited by the following article:

• TITLE: Sanger Sequencing for Molecular Diagnosis of SARS-CoV-2 Omicron Subvariants and Its Challenges

  AUTHORS: Sin Hang Lee

  KEYWORDS: eCDC, WHO, Sanger Sequencing, Omicron Variant, Minor Subvariants, BA.4/BA.5, L84I, BA.2, Multi-Allelic, Recombinant

  JOURNAL NAME: Journal of Biosciences and Medicines, Vol.10 No.9, September 21, 2022

  ABSTRACT: Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The European Centre for Disease Prevention and Control (eCDC) and the World Health Organization (WHO) recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the polymerase chain reaction (PCR)-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490 - 509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariants when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.2 RBD and an atypical BA.1 NTD, which can only be detected by using specially designed PCR primers. In addition, Sanger sequencing may reveal unclassified subvariants, such as BA.4/BA.5 with L84I mutation in the S gene NTD. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.