A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern

Abstract

The global spread of new SARS-CoV-2 variants of concern underscore an urgent need of simple deployed molecular tools that can differentiate these lineages. Several tools and protocols have been shared since the beginning of the COVID-19 pandemic, but they need to be timely adapted to cope with SARS-CoV-2 evolution. Although whole-genome sequencing (WGS) of the virus genetic material has been widely used, it still presents practical difficulties such as high cost, shortage of available reagents in the global market, need of a specialized laboratorial infrastructure and well-trained staff. These limitations result in SARS-CoV-2 surveillance blackouts across several countries. Here we propose a rapid and accessible protocol based on Sanger sequencing of a single PCR fragment that is able to identify and discriminate all SARS-CoV-2 variants of concern (VOCs) identified so far, according to each characteristic mutational profile at the Spike-RBD region (K417N/T, E484K, N501Y, A570D). Twelve COVID-19 samples from Brazilian patients were evaluated for both WGS and Sanger sequencing: three P.2, two P.1, six B.1.1 and one B.1.1.117 lineage. All results from the Sanger sequencing method perfectly matched the mutational profile of VOCs and non-VOCs RBD's characterized by WGS. In summary, this approach allows a much broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOCs and report results within a shorter time frame, which is of utmost importance in the context of rapid public health decisions in a fast evolving worldwide pandemic.

Keywords: Molecular surveillance; SARS-CoV-2 variants of concern; Sanger sequencing.

Fig. 1

Identification of Sars CoV-2 Spike-RBD mutations using Sanger sequencing. Commonly found RBD mutations flanked by the primer set (nucleotide positions from 22,797 to 23,522 at the Wu-1 genome) used for sequencing, including key mutations to enable identifying variants of concern and interest (A). 725 bp PCR fragments amplified from Sars Cov-2 cDNA (B). Sections from the electropherograms obtained by Sanger sequencing showing the E484K and N501Y VOC-associated mutations (C).