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Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima

This study aimed to exercise the Sanger sequencing strategy for screening of variants among confirmed COVID-19 cases and validate our strategy against NGS strains in Hiroshima retrieved from GISAID. A total of 660 samples from confirmed COVID-19 cases underwent screening for variants by Sanger-based...

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Autores principales: Ko, Ko, Takahashi, Kazuaki, Nagashima, Shintaro, E, Bunthen, Ouoba, Serge, Takafuta, Toshiro, Fujii, Yoshiki, Mimori, Michi, Okada, Fumie, Kishita, Eisaku, Ariyoshi, Kunie, Hussain, Md Razeen Ashraf, Sugiyama, Aya, Akita, Tomoyuki, Kuwabara, Masao, Tanaka, Junko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9030034/
https://www.ncbi.nlm.nih.gov/pubmed/35458450
http://dx.doi.org/10.3390/v14040720
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author Ko, Ko
Takahashi, Kazuaki
Nagashima, Shintaro
E, Bunthen
Ouoba, Serge
Takafuta, Toshiro
Fujii, Yoshiki
Mimori, Michi
Okada, Fumie
Kishita, Eisaku
Ariyoshi, Kunie
Hussain, Md Razeen Ashraf
Sugiyama, Aya
Akita, Tomoyuki
Kuwabara, Masao
Tanaka, Junko
author_facet Ko, Ko
Takahashi, Kazuaki
Nagashima, Shintaro
E, Bunthen
Ouoba, Serge
Takafuta, Toshiro
Fujii, Yoshiki
Mimori, Michi
Okada, Fumie
Kishita, Eisaku
Ariyoshi, Kunie
Hussain, Md Razeen Ashraf
Sugiyama, Aya
Akita, Tomoyuki
Kuwabara, Masao
Tanaka, Junko
author_sort Ko, Ko
collection PubMed
description This study aimed to exercise the Sanger sequencing strategy for screening of variants among confirmed COVID-19 cases and validate our strategy against NGS strains in Hiroshima retrieved from GISAID. A total of 660 samples from confirmed COVID-19 cases underwent screening for variants by Sanger-based partial sequencing to the targeted spike gene (nt22,735~nt23,532) using an in-house-developed primer set. The identification of variants was done by unique checkpoints of base nucleotide changes in the targeted spike gene. Moreover, we amplified one full-length genome using Sanger method and an in-house-developed primer library. Using NGS strains of the same sampling period from GISAID, a phylogenetic tree was constructed to examine the distribution pattern of variants in Hiroshima and to validate our Sanger method. The modified primer set provided 100% validation and 99.2% amplification. PANGO Lineage R.1 was detected in late in the third wave, followed by Alpha (B.1.1.7) domination in the fourth wave, Delta (B.1.617.2) domination in the fifth wave, and Omicron (B.1.1.529) domination in the sixth wave, and there was no significant difference in viral copies between variants (p = 0.09). The variants showed different transmission patterns, but the distribution of variants is consistent to that shown by the phylogenetic tree. The Sanger method also provided successful amplification of the full-length genome of the SARS-CoV-2 virus. Our Sanger sequencing strategy was useful for the screening of SASR-CoV-2 variants without the need for full-genome amplification. The modified primer set was validated to use universally, which allows an understanding of the variants’ distribution in real time and provides the evidence for policy-making and the formulation or modification of preventive strategies.
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spelling pubmed-90300342022-04-23 Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima Ko, Ko Takahashi, Kazuaki Nagashima, Shintaro E, Bunthen Ouoba, Serge Takafuta, Toshiro Fujii, Yoshiki Mimori, Michi Okada, Fumie Kishita, Eisaku Ariyoshi, Kunie Hussain, Md Razeen Ashraf Sugiyama, Aya Akita, Tomoyuki Kuwabara, Masao Tanaka, Junko Viruses Article This study aimed to exercise the Sanger sequencing strategy for screening of variants among confirmed COVID-19 cases and validate our strategy against NGS strains in Hiroshima retrieved from GISAID. A total of 660 samples from confirmed COVID-19 cases underwent screening for variants by Sanger-based partial sequencing to the targeted spike gene (nt22,735~nt23,532) using an in-house-developed primer set. The identification of variants was done by unique checkpoints of base nucleotide changes in the targeted spike gene. Moreover, we amplified one full-length genome using Sanger method and an in-house-developed primer library. Using NGS strains of the same sampling period from GISAID, a phylogenetic tree was constructed to examine the distribution pattern of variants in Hiroshima and to validate our Sanger method. The modified primer set provided 100% validation and 99.2% amplification. PANGO Lineage R.1 was detected in late in the third wave, followed by Alpha (B.1.1.7) domination in the fourth wave, Delta (B.1.617.2) domination in the fifth wave, and Omicron (B.1.1.529) domination in the sixth wave, and there was no significant difference in viral copies between variants (p = 0.09). The variants showed different transmission patterns, but the distribution of variants is consistent to that shown by the phylogenetic tree. The Sanger method also provided successful amplification of the full-length genome of the SARS-CoV-2 virus. Our Sanger sequencing strategy was useful for the screening of SASR-CoV-2 variants without the need for full-genome amplification. The modified primer set was validated to use universally, which allows an understanding of the variants’ distribution in real time and provides the evidence for policy-making and the formulation or modification of preventive strategies. MDPI 2022-03-30 /pmc/articles/PMC9030034/ /pubmed/35458450 http://dx.doi.org/10.3390/v14040720 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ko, Ko
Takahashi, Kazuaki
Nagashima, Shintaro
E, Bunthen
Ouoba, Serge
Takafuta, Toshiro
Fujii, Yoshiki
Mimori, Michi
Okada, Fumie
Kishita, Eisaku
Ariyoshi, Kunie
Hussain, Md Razeen Ashraf
Sugiyama, Aya
Akita, Tomoyuki
Kuwabara, Masao
Tanaka, Junko
Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima
title Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima
title_full Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima
title_fullStr Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima
title_full_unstemmed Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima
title_short Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima
title_sort exercising the sanger sequencing strategy for variants screening and full-length genome of sars-cov-2 virus during alpha, delta, and omicron outbreaks in hiroshima
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9030034/
https://www.ncbi.nlm.nih.gov/pubmed/35458450
http://dx.doi.org/10.3390/v14040720
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