A Simplified Sanger Sequencing Method for Detection of Relevant SARS-CoV-2 Variants

Abstract

Molecular surveillance of the new coronavirus through new genomic sequencing technologies revealed the circulation of important variants of SARS-CoV-2. Sanger sequencing has been useful in identifying important variants of SARS-CoV-2 without the need for whole-genome sequencing. A sequencing protocol was constructed to cover a region of 1000 base pairs, from a 1120 bp product generated after a two-step RT-PCR assay in samples positive for SARS-CoV-2. Consensus sequence construction and mutation identification were performed. Of all 103 samples sequenced, 69 contained relevant variants represented by 20 BA.1, 13 delta, 22 gamma, and 14 zeta, identified between June 2020 and February 2022. All sequences found were aligned with representative sequences of the variants. Using the Sanger sequencing methodology, we were able to develop a more accessible protocol to assist viral surveillance with a more accessible platform.

Keywords: SARS-CoV-2; Sanger; surveillance; variants.

Figure 1

Distribution of studied variants in 2020 to 2022. Distribution of the variants detected in the study period, by month: wild type (B.1) as red, zeta (P.2) as blue, gamma (P.1) as green, delta (B.1.617.2) as yellow, omicron (BA.1) as purple. It can be observed that the B.1 strains and the P.1 variant had the highest prevalence among the analyzed samples, while the BA.1 was the only variant found in the first two months of 2022.

Figure 2

Primer placement and detection coverage. Positioning of the amplification and sequencing primers within the S gene. The positioning of the primers allows identification of the 25 mutations signaled in the image. The first round of PCR (primer forward 75 and primer reverse 79) amplifies an extensive region of the gene, covering the entire portion of RBD up to the initial portion of the S2 subunit. The product of the second round (Primer Forward CoV-17 and Primer Reverse 78) delimits the region for sequencing and identification of mutations.

Figure 3

Alignment of the sequences with the representative of each variant and the reference generated in Wuhan. Protein sequences of some samples were aligned with templates for zeta (P.2), gamma (P.1), delta (B.1.617.2), and omicron (B.1.1.529). BA.1 plus the reference sequence generated in Wuhan. This alignment was done in the BioEdit program. The mutation profile found in each sample was paired with the one found in the template samples, confirming the classification adopted for the samples (SCOV-1627, SCOV-1108—zeta; SCOV-2055, SCOV-1826, SCOV-2056—gamma; SCOV-2154, SCOV-2732, SCOV-2731—delta; SCOV-2842, SCOV-2846, SCOV-3440—omicron). The illustrated protein alignment corresponds to position 417 to 705 of the spike.