Mass Screening of SARS-CoV-2 Variants using Sanger Sequencing Strategy in Hiroshima, Japan

Abstract

This study aimed to develop the feasible and effective universal screening strategy of the notable SARS-CoV-2 variants by Sanger Sequencing Strategy and then practically applied it for mass screening in Hiroshima, Japan. A total of 734 samples from COVID-19 confirmed cases in Hiroshima were screened for the notable SARS-CoV-2 variants (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, C.37, B.1.1.529, etc.). The targeted spike region is amplified by nested RT-PCR using in-house designed primer set hCoV-Spike-A and standard amplification protocol. Additionally, randomly selected 96 samples were also amplified using primer sets hCoV-Spike-B and hCoV-Spike-C. The negative amplified samples were repeated for second attempt of amplification by volume-up protocol. Thereafter, the amplified products were assigned for Sanger sequencing using corresponding primers. The positive amplification rate of primer set hCoV-Spike-A, hCoV-Spike-B and hCoV-Spike-C were 87.3%, 83.3% and 93.8% respectively for standard protocol and increased to 99.6%, 95.8% and 96.9% after second attempt by volume-up protocol. The readiness of genome sequences was 96.9%, 100% and 100% respectively. Among 48 mutant isolates, 26 were B.1.1.7 (Alpha), 7 were E484K single mutation and the rest were other types of mutation. Moreover, 5 cluster cases with single mutation at N501S were firstly reported in Hiroshima. This study indicates the reliability and effectiveness of Sanger sequencing to screen large number of samples for the notable SARS-CoV-2 variants. Compared to the Next Generation Sequencing (NGS), our method introduces the feasible, universally applicable, and practically useful tool for identification of the emerging variants with less expensive and time consuming especially in those countries where the NGS is not practically available. Our method allows not only to identify the pre-existing variants but also to examine other rare type of mutation or newly emerged variants and is crucial for prevention and control of pandemic.

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

“…It has been considered a VOC due to its high transmissibility, short incubation period, and potential evasion of the neutralizing activity of antibodies generated by vaccine and previous infections [ 7 , 32 ]. In these samples, we identified three delta-specific mutations (L452R, T478K, and P681R) in addition to D614G, in agreement with other sequencing protocols, that used the same technology, capable of identifying delta [ 33 ].…”

Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger

“…Recently, Sanger sequencing methodologies that study the S protein have been published and even standardised commercial protocols are already available 28 , 29 , 30 . Several of those studies require a larger number of primers targeting domains of interest, such as receptor-binding domains and receptor-binding motifs 30 , as well as analysing mutations in other regions 28 , and in some cases they perform a total sequencing of the S protein. All these previous methodologies involve performing multiple PCRs 29 , which increases the cost compared to the methodology that we propose here.…”

A full genome tiling array enhanced the inspection and quarantine of SARS-CoV-2

“…It can also provide genome sequences and mutations of the virus, which is of great significance for virus traceability, virulence, and vaccine evaluation. Sanger sequencing [ 18 ] and Next-generation sequencing (NGS) are mature technology at present. The WHO has recommended that countries sequence at least 1% of their SARS-CoV-2 positive samples to detect emerging VOCs and significant mutations in the virus.…”