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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.

SUBMITTER: Deminco F

PROVIDER: S-EPMC9689870 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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

Deminco Felice F Vaz Sara N SN Santana Daniele S DS Pedroso Celia C Tadeu Jean J Stoecker Andreas A Vieira Sueli M SM Netto Eduardo E Brites Carlos C

Diagnostics (Basel, Switzerland) 20221027 11

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 ...[more]

PMID: 36359452

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Project description: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.

Project description:BackgroundThe evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists, giving rise to new variants characterized by mutations in the spike protein. However, public data regarding the virus's evolutionary trend is not widely available after the downgrade of coronavirus disease 2019(COVID-19). Therefore, this study aimed to investigate the applicability of an in-house Sanger-based method for identifying SARS-CoV-2 variants, particularly focusing on newly emerged Omicron variants, and updating the epidemiology of COVID-19 during the 8th wave in Hiroshima Prefecture.ResultsA total of 639 saliva samples of individuals who had tested positive for COVID-19, received from Hiroshima City Medical Association Clinical Laboratory Center between February 01, 2023, and March 12, 2024, were included in the study. SARS-CoV-2 variants were identified in 69.3% (443/639) with the mean viral titer 2 × 106 copies/mL, and high viral titer in Omicron variant XBC.1.6* (5 × 108 copies/mL) using RT-qPCR. By partial Spike gene-based sequencing using the Sanger Sequencing strategy, Omicron sub-lineages XXB.1, BA.5, and EG.1 were identified during different periods. A comprehensive phylogenetic analysis of 7383 SARS-CoV-2 strains retrieved from GISAID, collected in Hiroshima from the onset of the COVID-19 pandemic in early 2020 until July 2024, revealed the dynamic evolution of SARS-CoV-2 variants over time. The study found a similar pattern of variant distribution between the full genomes from GISAID, and the partial genomes obtained from our screening strategy during the same period.ConclusionsOur study revealed that all SARS-CoV-2 viruses circulated in Hiroshima were Omicron variants and their sub-lineages during the 8th wave outbreak in Hiroshima. Persistent molecular surveillance of SARS-CoV-2 is needed for the decision-making and strategic planning of the public promptly. Our study added evidence for the usefulness of SARS-CoV-2 spike gene partial sequencing-based SARS-CoV-2 variant identification strategy for mass screening and molecular surveillance even though the evolution of newly emerged various SARS-CoV-2 Omicron variants.

Dataset Information

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

Publications

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

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