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COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens

With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for...

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Autores principales: Sugi, Tatsuki, Rahman, Mizanur, Rahim, Rummana, Hasan, Abu, Kawai, Naoko, Hayashida, Kyoko, Yamagishi, Junya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9534710/
https://www.ncbi.nlm.nih.gov/pubmed/36212105
http://dx.doi.org/10.1155/2022/2109641
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author Sugi, Tatsuki
Rahman, Mizanur
Rahim, Rummana
Hasan, Abu
Kawai, Naoko
Hayashida, Kyoko
Yamagishi, Junya
author_facet Sugi, Tatsuki
Rahman, Mizanur
Rahim, Rummana
Hasan, Abu
Kawai, Naoko
Hayashida, Kyoko
Yamagishi, Junya
author_sort Sugi, Tatsuki
collection PubMed
description With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for whole-genome resequencing, which can make the pipeline robust for mutations at the primer annealing site by a redundant amplicon scheme. We further demonstrated that subtracting overrepresented amplicons from the multiplex PCR products reduced the bias of the next-generation sequencing (NGS) library, resulting in decreasing required sequencing reads per sample. We applied this sequencing strategy to clinical specimens collected in Bangladesh. More than 80% out of the 304 samples were successfully sequenced. Less than 5% were ambiguous nucleotides, and several known variants were detected. With the additional strategies presented here, we believe that whole-genome resequencing of SARS-CoV-2 from clinical samples can be optimized.
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spelling pubmed-95347102022-10-06 COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens Sugi, Tatsuki Rahman, Mizanur Rahim, Rummana Hasan, Abu Kawai, Naoko Hayashida, Kyoko Yamagishi, Junya Interdiscip Perspect Infect Dis Research Article With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for whole-genome resequencing, which can make the pipeline robust for mutations at the primer annealing site by a redundant amplicon scheme. We further demonstrated that subtracting overrepresented amplicons from the multiplex PCR products reduced the bias of the next-generation sequencing (NGS) library, resulting in decreasing required sequencing reads per sample. We applied this sequencing strategy to clinical specimens collected in Bangladesh. More than 80% out of the 304 samples were successfully sequenced. Less than 5% were ambiguous nucleotides, and several known variants were detected. With the additional strategies presented here, we believe that whole-genome resequencing of SARS-CoV-2 from clinical samples can be optimized. Hindawi 2022-09-28 /pmc/articles/PMC9534710/ /pubmed/36212105 http://dx.doi.org/10.1155/2022/2109641 Text en Copyright © 2022 Tatsuki Sugi et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Sugi, Tatsuki
Rahman, Mizanur
Rahim, Rummana
Hasan, Abu
Kawai, Naoko
Hayashida, Kyoko
Yamagishi, Junya
COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens
title COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens
title_full COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens
title_fullStr COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens
title_full_unstemmed COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens
title_short COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens
title_sort covid-19 whole-genome resequencing with redundant tiling pcr and subtract-based amplicon normalization successfully characterized sars-cov-2 variants in clinical specimens
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9534710/
https://www.ncbi.nlm.nih.gov/pubmed/36212105
http://dx.doi.org/10.1155/2022/2109641
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