![]() Altogether, 12 mutations were detected in the receptor-binding motif across the 20 samples. This sample lacks G446S, L452R, E484K, and G496S mutations and is identified as Omicron BA.2 (B.1.1.529+BA.2) ( Fig. 3 Fig. One nasal swab sample received on 5 February 2022 showed eight mutations including N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y505H. These samples lack L452R and E484K mutations and are identified as Omicron BA.1 (B.1.1.529+BA.1). Sequence alignment of the 273-bp amplicon revealed 10 mutations including N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H in six nasal swab samples. Based on the presence or absence of a specific mutation(s), samples are designated under Alpha, Beta/Gamma, or Delta variants ( Fig. 2 Fig. Sequence alignment of the 246-bp amplicon revealed five different mutations in 13 saliva samples: L452R, T478K, E484K, Q493R, and N501Y ( Fig. 2). The reverse-complemented sequencing data were used for sequence alignment and mutation analysis against SARS-CoV-2 Wuhan reference sequence NC_045512. The use of a reverse primer for sequencing covered more mutation areas present in Sars-CoV-2 variants. Sanger sequencing from a gel-extracted PCR amplicon provided a clean chromatogram for reliable data analysis (Fig. Based upon unique mutation signatures obtained from whole-genome sequence alignments of different SARS-CoV-2 variants and literature (WHO and CDC), shorter segments (<275 bp) of the spike protein receptor-binding domain were targeted for Sanger sequencing to identify variants quickly ( Fig. 1). Sanger sequencing can provide a larger read (~1.2 kb) to target a single amplicon of 0.5 to 1 kb, but samples such as saliva, nasal swabs, or even wastewater often lack intact RNA segments, making it challenging to generate larger amplicons in reverse transcriptase quantitative PCR (RT-qPCR)-based approaches. Once the whole genome of a new variant is characterized, single-amplicon-based Sanger sequencing of a targeted viral genome segment is a cost-effective and quick alternative to variant tracking ( 19). In many countries or areas worldwide, lack of instrumentation, reagent facilities, data storage issues, bioinformatics support, and time requirements limit the usage of WGS for routine clinical use or surveillance worldwide. However, NGS that can generate thousands of reads per sequence in parallel is rapidly adapted in sequencing the whole genome of SARS-CoV-2 and variant identifications ( 17, 18). Both Sanger sequencing and next-generation sequencing (NGS) were combined to characterize the first whole genome of SARS-CoV-2 from a 2019-nCoV patient sample ( 16). Sanger sequencing was utilized in a few instances for whole-genome sequencing of SARS-CoV-2 ( 14, 15). Whole-genome sequencing (WGS) is the gold standard for accurately characterizing new viral genomes and variant designations ( 11 – 13). Monitoring mutational changes and tracking emerging variants on time are critical to modifying vaccine booster strategies and new therapeutic development. However, the emerging Omicron variant bearing 30 mutations in the spike protein with 15 amino acid substitutions in the receptor-binding domain (RBD) has raised the alarm of reduced vaccine ( 8) or monoclonal antibody therapy ( 9, 10) efficacy. Preventive vaccination ( 4 – 6) and therapeutic strategies ( 7) have been effective against past variants of concern. The World Health Organization (WHO) and the United States Centers for Disease Control and Prevention (CDC) classified the past and present variants of concern (VOC), demonstrating the frequency of viral mutation ( 2, 3). Frequent viral mutation and new variant formation have delayed the end of the pandemic. The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in 5.9 million deaths from December 2019 to February 2022 ( 1). ![]()
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