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.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

“…The ethics approval was issued on June 12, 2020. SARS-CoV-2 VOCs and VOIs included in this study were isolated from symptomatic patients (Ct value < 25, using E gene as target on RT-qPCR—Kit Molecular SARS-CoV-2 E/RP Bio-Manguinhos Fiocruz), in the State Pernambuco, Northeast Brazil ( Bezerra et al, 2021 ; Supplementary Table 3 ). The study was approved by the local Human Research Ethics Committee (CAAE: 32333120.4.0000.5190).…”

SARS-CoV-2 variant detection with ADSSpike

“…In contrast to WGS, amplicon deep sequencing focuses on a specific gene or genomic region, providing a greater sequencing depth and coverage while reducing costs and data burden. Sanger-based approaches have been previously employed ( Bezerra et al, 2021 , Goes et al, 2021 ). These are either focused on a small window in the S gene, and hence missing relevant SNPs flanking said region, or target SNPs prevalent to specific VOC/VOIs.…”

Different Strategies for the Identification of SARS-CoV-2 Variants in the Laboratory Practice

“…It should be mentioned that in the case of a large number of samples, the costs of NGS sequencing are dramatically reduced and can be competitive with Sanger sequencing. In these cases, the use of this latter method should be limited to confirmation of mutations within the S gene when a limited number of cases are analysed [ 17 ].…”