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Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design
BACKGROUND: As the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive. OBJ...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Nature Singapore
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9160177/ https://www.ncbi.nlm.nih.gov/pubmed/35653026 http://dx.doi.org/10.1007/s13258-022-01264-7 |
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author | Jeong, Haeyoung Lee, Siseok Ko, Junsang Ko, Minsu Seo, Hwi Won |
author_facet | Jeong, Haeyoung Lee, Siseok Ko, Junsang Ko, Minsu Seo, Hwi Won |
author_sort | Jeong, Haeyoung |
collection | PubMed |
description | BACKGROUND: As the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive. OBJECTIVE: To extract conserved sequences from SARS-CoV-2 genomes for the design of diagnostic PCR primers using a bioinformatics approach that can handle massive genomic sequences efficiently. METHODS: A total of 230,163 full-length viral genomes were retrieved from the NCBI SARS-CoV-2 Resources and GISAID EpiCoV database. This number was reduced to 14.11% following removal of 5′-/3′-untranslated regions and sequence dereplication. Fast, reference-based, multiple sequence alignments identified conserved sequences and specific primer sets were designed against these regions using a conventional tool. Primer sets chosen among the candidates were evaluated by in silico PCR and RT-qPCR. RESULTS: Out of 17 conserved sequences (totaling 4.3 kb), two primer sets targeting the nsp2 and ORF3a genes were picked that exhibited > 99.9% in silico amplification coverage against the original dataset (230,163 genomes) when a 5% mismatch between the primers and target was allowed. In addition, the primer sets successfully detected nine SARS-CoV-2 variant RNA samples (Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa) in experimental RT-qPCR validations. CONCLUSION: In addition to the RdRp, E, N, and S genes that are targeted commonly, our approach can be used to identify novel primer targets in SARS-CoV-2 and should be a priority strategy in the event of novel SARS-CoV-2 variants or other pandemic outbreaks. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13258-022-01264-7. |
format | Online Article Text |
id | pubmed-9160177 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Springer Nature Singapore |
record_format | MEDLINE/PubMed |
spelling | pubmed-91601772022-06-02 Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design Jeong, Haeyoung Lee, Siseok Ko, Junsang Ko, Minsu Seo, Hwi Won Genes Genomics Research Article BACKGROUND: As the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive. OBJECTIVE: To extract conserved sequences from SARS-CoV-2 genomes for the design of diagnostic PCR primers using a bioinformatics approach that can handle massive genomic sequences efficiently. METHODS: A total of 230,163 full-length viral genomes were retrieved from the NCBI SARS-CoV-2 Resources and GISAID EpiCoV database. This number was reduced to 14.11% following removal of 5′-/3′-untranslated regions and sequence dereplication. Fast, reference-based, multiple sequence alignments identified conserved sequences and specific primer sets were designed against these regions using a conventional tool. Primer sets chosen among the candidates were evaluated by in silico PCR and RT-qPCR. RESULTS: Out of 17 conserved sequences (totaling 4.3 kb), two primer sets targeting the nsp2 and ORF3a genes were picked that exhibited > 99.9% in silico amplification coverage against the original dataset (230,163 genomes) when a 5% mismatch between the primers and target was allowed. In addition, the primer sets successfully detected nine SARS-CoV-2 variant RNA samples (Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa) in experimental RT-qPCR validations. CONCLUSION: In addition to the RdRp, E, N, and S genes that are targeted commonly, our approach can be used to identify novel primer targets in SARS-CoV-2 and should be a priority strategy in the event of novel SARS-CoV-2 variants or other pandemic outbreaks. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13258-022-01264-7. Springer Nature Singapore 2022-06-02 2022 /pmc/articles/PMC9160177/ /pubmed/35653026 http://dx.doi.org/10.1007/s13258-022-01264-7 Text en © The Author(s) under exclusive licence to The Genetics Society of Korea 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. |
spellingShingle | Research Article Jeong, Haeyoung Lee, Siseok Ko, Junsang Ko, Minsu Seo, Hwi Won Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design |
title | Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design |
title_full | Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design |
title_fullStr | Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design |
title_full_unstemmed | Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design |
title_short | Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design |
title_sort | identification of conserved regions from 230,163 sars-cov-2 genomes and their use in diagnostic pcr primer design |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9160177/ https://www.ncbi.nlm.nih.gov/pubmed/35653026 http://dx.doi.org/10.1007/s13258-022-01264-7 |
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