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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...
Autores principales: | , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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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. |
format | Online Article Text |
id | pubmed-9030034 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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