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High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination

For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover,...

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Autores principales: Liao, Yu-Chieh, Chen, Feng-Jui, Chuang, Min-Chieh, Wu, Han-Chieh, Ji, Wan-Chen, Yu, Guann-Yi, Huang, Tsi-Shu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8954144/
https://www.ncbi.nlm.nih.gov/pubmed/35328676
http://dx.doi.org/10.3390/ijms23063257
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author Liao, Yu-Chieh
Chen, Feng-Jui
Chuang, Min-Chieh
Wu, Han-Chieh
Ji, Wan-Chen
Yu, Guann-Yi
Huang, Tsi-Shu
author_facet Liao, Yu-Chieh
Chen, Feng-Jui
Chuang, Min-Chieh
Wu, Han-Chieh
Ji, Wan-Chen
Yu, Guann-Yi
Huang, Tsi-Shu
author_sort Liao, Yu-Chieh
collection PubMed
description For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in two separate PCRs to obtain spike gene sequences. To overcome these disadvantages, we proposed a single PCR to efficiently detect spike gene mutations. We proposed a bioinformatic protocol that can process FASTQ reads into spike gene consensus sequences to accurately call spike protein variants from sequenced samples or to fairly express the cases of missing amplicons. We evaluated the in silico detection rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in silico detection rate of our proposed single PCR primers was 97.07%. We demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of spike genes for variant SARS-CoV-2 determination. Our protocol works well with the data yielded from versatile primer designs, making it easy to determine spike protein variants.
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spelling pubmed-89541442022-03-26 High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination Liao, Yu-Chieh Chen, Feng-Jui Chuang, Min-Chieh Wu, Han-Chieh Ji, Wan-Chen Yu, Guann-Yi Huang, Tsi-Shu Int J Mol Sci Article For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in two separate PCRs to obtain spike gene sequences. To overcome these disadvantages, we proposed a single PCR to efficiently detect spike gene mutations. We proposed a bioinformatic protocol that can process FASTQ reads into spike gene consensus sequences to accurately call spike protein variants from sequenced samples or to fairly express the cases of missing amplicons. We evaluated the in silico detection rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in silico detection rate of our proposed single PCR primers was 97.07%. We demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of spike genes for variant SARS-CoV-2 determination. Our protocol works well with the data yielded from versatile primer designs, making it easy to determine spike protein variants. MDPI 2022-03-17 /pmc/articles/PMC8954144/ /pubmed/35328676 http://dx.doi.org/10.3390/ijms23063257 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
Liao, Yu-Chieh
Chen, Feng-Jui
Chuang, Min-Chieh
Wu, Han-Chieh
Ji, Wan-Chen
Yu, Guann-Yi
Huang, Tsi-Shu
High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination
title High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination
title_full High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination
title_fullStr High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination
title_full_unstemmed High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination
title_short High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination
title_sort high-integrity sequencing of spike gene for sars-cov-2 variant determination
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8954144/
https://www.ncbi.nlm.nih.gov/pubmed/35328676
http://dx.doi.org/10.3390/ijms23063257
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