Tracking SARS-CoV-2 variants by entire S-gene analysis using long-range RT-PCR and Sanger sequencing

Abstract

Introduction: Genomic surveillance of the SARS-CoV-2 virus is important to assess transmissibility, disease severity, and vaccine effectiveness. The SARS-CoV-2 genome consists of approximately 30 kb single-stranded RNA that is too large to analyze the whole genome by Sanger sequencing. Thus, in this study, we performed Sanger sequencing following long-range RT-PCR of the entire SARS-CoV-2 S-gene and analyzed the mutational dynamics.

Methods: The 4 kb region, including the S-gene, was amplified by two-step long-range RT-PCR. Then, the entire S-gene sequence was determined by Sanger sequencing. The amino acid mutations were identified as compared with the reference SARS-CoV-2 genome.

Results: The S:D614G mutation was found in all samples. The R.1 variants were detected after January 2021. Alpha variants started to emerge in April 2021. Delta variants replaced Alpha in July 2021. Then, Omicron variants were detected after December 2021. These mutational dynamics in samples collected in the Chiba University Hospital were similar to those in Japan.

Conclusion: The emergence of variants of concern (VOC) has been reported by the entire S-gene analysis. As the VOCs have unique mutational patterns of the S-gene region, analysis of the entire S-gene will be useful for molecular surveillance of the SARS-CoV-2 in clinical laboratories.

Keywords: Long-range RT-PCR; S-gene; SARS-CoV-2; Sanger sequencing; Surveillance.

Fig. 1

Method overview of the long-range RT-PCR followed by Sanger sequencing. The SARS-CoV-2 genome consists of 29,903 bases (blue line). The S-gene is encoded 21,563–25,384 position of the genome (blue box). “RT-PCR,” dotted line and solid lines indicate first-strand cDNA and following PCR amplification region, respectively. Sequencing, dashed lines indicate sequencing regions. Triangles indicate primer binding sites.

Fig. 2

Correlations of viral RNA concentration, the success rate of sequencing, and the Cq values of long-range RT-PCR. (A) viral RNA concentration vs. success rate of sequencing. Quartile groups: Q1 (≤3.0 log10 copies/uL), Q2 (>3.0–≤4.0 log10 copies/uL), Q3 (>4.0–≤5.0 log10 copies/uL), and Q4 (>5.0 log10 copies/uL). The success rate of sequencing: Q1, 68% (27/40); Q2, 81% (39/48); Q3, 94% (46/49); and Q4, 100% (46/46). (B) viral RNA concentration vs. Cq values of long-range RT-PCR. The Cq values of long-range RT-PCR were negatively correlated with viral RNA concentration. The p-value was determined by Spearman rank-order correlation test.

Fig. 3

The mutational pattern of spike protein among the frequently observed SARS-CoV-2 variants in Japan.

Fig. 4

Tracking SARS-CoV-2 variants by the spike protein mutation. “Undetermined” includes various lineages of SARS-CoV-2 which could not be determined the lineages because of the lack of specific mutation patterns except for S:D614G.