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Rapid whole genome sequencing methods for RNA viruses

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome seq...

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Autores principales: Misu, Masayasu, Yoshikawa, Tomoki, Sugimoto, Satoko, Takamatsu, Yuki, Kurosu, Takeshi, Ouji, Yukiteru, Yoshikawa, Masahide, Shimojima, Masayuki, Ebihara, Hideki, Saijo, Masayuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9995502/
https://www.ncbi.nlm.nih.gov/pubmed/36910229
http://dx.doi.org/10.3389/fmicb.2023.1137086
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author Misu, Masayasu
Yoshikawa, Tomoki
Sugimoto, Satoko
Takamatsu, Yuki
Kurosu, Takeshi
Ouji, Yukiteru
Yoshikawa, Masahide
Shimojima, Masayuki
Ebihara, Hideki
Saijo, Masayuki
author_facet Misu, Masayasu
Yoshikawa, Tomoki
Sugimoto, Satoko
Takamatsu, Yuki
Kurosu, Takeshi
Ouji, Yukiteru
Yoshikawa, Masahide
Shimojima, Masayuki
Ebihara, Hideki
Saijo, Masayuki
author_sort Misu, Masayasu
collection PubMed
description RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5′ and 3′ terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences’ accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.
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spelling pubmed-99955022023-03-10 Rapid whole genome sequencing methods for RNA viruses Misu, Masayasu Yoshikawa, Tomoki Sugimoto, Satoko Takamatsu, Yuki Kurosu, Takeshi Ouji, Yukiteru Yoshikawa, Masahide Shimojima, Masayuki Ebihara, Hideki Saijo, Masayuki Front Microbiol Microbiology RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5′ and 3′ terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences’ accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes. Frontiers Media S.A. 2023-02-23 /pmc/articles/PMC9995502/ /pubmed/36910229 http://dx.doi.org/10.3389/fmicb.2023.1137086 Text en Copyright © 2023 Misu, Yoshikawa, Sugimoto, Takamatsu, Kurosu, Ouji, Yoshikawa, Shimojima, Ebihara and Saijo. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Misu, Masayasu
Yoshikawa, Tomoki
Sugimoto, Satoko
Takamatsu, Yuki
Kurosu, Takeshi
Ouji, Yukiteru
Yoshikawa, Masahide
Shimojima, Masayuki
Ebihara, Hideki
Saijo, Masayuki
Rapid whole genome sequencing methods for RNA viruses
title Rapid whole genome sequencing methods for RNA viruses
title_full Rapid whole genome sequencing methods for RNA viruses
title_fullStr Rapid whole genome sequencing methods for RNA viruses
title_full_unstemmed Rapid whole genome sequencing methods for RNA viruses
title_short Rapid whole genome sequencing methods for RNA viruses
title_sort rapid whole genome sequencing methods for rna viruses
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9995502/
https://www.ncbi.nlm.nih.gov/pubmed/36910229
http://dx.doi.org/10.3389/fmicb.2023.1137086
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