In-House, Rapid, and Low-Cost SARS-CoV-2 Spike Gene Sequencing Protocol to Identify Variants of Concern Using Sanger Sequencing

Abstract

Background: The need for active genomic sequencing surveillance to rapidly identify circulating SARS-CoV-2 variants of concern (VOCs) is critical. However, increased global demand has led to a shortage of commercial SARS-CoV-2 sequencing kits, and not every country has the technological capability or the funds for high-throughput sequencing platforms. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol that uses supplies, equipment, and methodologic expertise available in standard molecular or diagnostic laboratories to identify circulating SARS-CoV-2 variants of concern. Methods: Sets of primers flanking the SARS-CoV-2 spike gene were designed using SARS-CoV-2 genome sequences retrieved from the Global Initiative on Sharing Avian Influenza Data (GISAID) Database and synthesized in-house. Primer specificity and final sequences were verified using online prediction analyses with BLAST. The primers were validated using 282 nasopharyngeal samples collected from patients assessed as positive for SARS-CoV-2 at the diagnostic laboratory of the hospital using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit. The patient samples were subjected to RNA extraction followed by cDNA synthesis, conventional polymerase chain reaction, and Sanger sequencing. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole genome sequencing of the same samples. Results: Sanger sequencing using the newly designed primers and next-generation whole genome sequencing of 282 patient samples indicated identical variants of concern results: 123 samples contained the alpha variant (B.1.1.7); 78, beta (B.1.351), 0, gamma (P.1), and 13, delta (B.1.617.2). Moreover, the remaining samples were non-VOC that belonged to none of these variants and had 99.97% identity with the reference genome. Only four samples had poor sequence quality by Sanger sequencing owing to a low viral count (Ct value >38). Therefore, mutation calls were >98% accurate. Conclusions: Sanger sequencing method using in-house primers is an alternative approach that can be used in facilities with existing equipment to mitigate limitations in high throughput supplies required to identify SARS-CoV-2 variants of concern during the COVID-19 pandemic. This protocol is easily adaptable for detection of emerging variants.