An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates

Autores
Goya, Stephanie; Valinotto, Laura Elena; Tittarelli, Estefanía; Rojo, Gabriel Lihue; Nabaes Jodar, Mercedes Soledad; Greninger, Alexander L.; Zaiat, Jonathan Javier; Marti, Marcelo Adrian; Mistchenko, Alicia Susana; Viegas, Mariana
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1–3 (HPIV1-3) were also obtained with the selected optimal methodology.
Fil: Goya, Stephanie. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Valinotto, Laura Elena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Tittarelli, Estefanía. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Rojo, Gabriel Lihue. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina
Fil: Nabaes Jodar, Mercedes Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Greninger, Alexander L.. University of Washington; Estados Unidos
Fil: Zaiat, Jonathan Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina
Fil: Viegas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina
Materia
Respiratory viruses
NGS
Respiratory syncytial virus
Viral complete genomes
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/97005
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/97005
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspiratesGoya, StephanieValinotto, Laura ElenaTittarelli, EstefaníaRojo, Gabriel LihueNabaes Jodar, Mercedes SoledadGreninger, Alexander L.Zaiat, Jonathan JavierMarti, Marcelo AdrianMistchenko, Alicia SusanaViegas, MarianaRespiratory virusesNGSRespiratory syncytial virusViral complete genomeshttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1–3 (HPIV1-3) were also obtained with the selected optimal methodology.Fil: Goya, Stephanie. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valinotto, Laura Elena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tittarelli, Estefanía. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rojo, Gabriel Lihue. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Nabaes Jodar, Mercedes Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Greninger, Alexander L.. University of Washington; Estados UnidosFil: Zaiat, Jonathan Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Viegas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaPublic Library of Science2018-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/97005Goya, Stephanie; Valinotto, Laura Elena; Tittarelli, Estefanía; Rojo, Gabriel Lihue; Nabaes Jodar, Mercedes Soledad; et al.; An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates; Public Library of Science; Plos One; 13; 6; 6-2018; 1-151932-6203CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0199714info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199714info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:11:34Zoai:ri.conicet.gov.ar:11336/97005instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-10 13:11:34.253CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
title An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
spellingShingle An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
Goya, Stephanie
Respiratory viruses
NGS
Respiratory syncytial virus
Viral complete genomes
title_short An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
title_full An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
title_fullStr An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
title_full_unstemmed An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
title_sort An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
dc.creator.none.fl_str_mv Goya, Stephanie
Valinotto, Laura Elena
Tittarelli, Estefanía
Rojo, Gabriel Lihue
Nabaes Jodar, Mercedes Soledad
Greninger, Alexander L.
Zaiat, Jonathan Javier
Marti, Marcelo Adrian
Mistchenko, Alicia Susana
Viegas, Mariana
author Goya, Stephanie
author_facet Goya, Stephanie
Valinotto, Laura Elena
Tittarelli, Estefanía
Rojo, Gabriel Lihue
Nabaes Jodar, Mercedes Soledad
Greninger, Alexander L.
Zaiat, Jonathan Javier
Marti, Marcelo Adrian
Mistchenko, Alicia Susana
Viegas, Mariana
author_role author
author2 Valinotto, Laura Elena
Tittarelli, Estefanía
Rojo, Gabriel Lihue
Nabaes Jodar, Mercedes Soledad
Greninger, Alexander L.
Zaiat, Jonathan Javier
Marti, Marcelo Adrian
Mistchenko, Alicia Susana
Viegas, Mariana
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Respiratory viruses
NGS
Respiratory syncytial virus
Viral complete genomes
topic Respiratory viruses
NGS
Respiratory syncytial virus
Viral complete genomes
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1–3 (HPIV1-3) were also obtained with the selected optimal methodology.
Fil: Goya, Stephanie. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Valinotto, Laura Elena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Tittarelli, Estefanía. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Rojo, Gabriel Lihue. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina
Fil: Nabaes Jodar, Mercedes Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Greninger, Alexander L.. University of Washington; Estados Unidos
Fil: Zaiat, Jonathan Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina
Fil: Viegas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina
description Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1–3 (HPIV1-3) were also obtained with the selected optimal methodology.
publishDate 2018
dc.date.none.fl_str_mv 2018-06
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/97005
Goya, Stephanie; Valinotto, Laura Elena; Tittarelli, Estefanía; Rojo, Gabriel Lihue; Nabaes Jodar, Mercedes Soledad; et al.; An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates; Public Library of Science; Plos One; 13; 6; 6-2018; 1-15
1932-6203
CONICET Digital
CONICET
url http://hdl.handle.net/11336/97005
identifier_str_mv Goya, Stephanie; Valinotto, Laura Elena; Tittarelli, Estefanía; Rojo, Gabriel Lihue; Nabaes Jodar, Mercedes Soledad; et al.; An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates; Public Library of Science; Plos One; 13; 6; 6-2018; 1-15
1932-6203
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0199714
info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199714
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
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dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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