Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R))
Autor(a) principal: | |
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Data de Publicação: | 2015 |
Outros Autores: | , , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://www.sciencedirect.com/science/article/pii/S0166093415001457 http://hdl.handle.net/11449/128604 |
Resumo: | Sequence-independent methods for viral discovery have been widely used for whole genome sequencing of viruses. Different protocols for viral enrichment, library preparation and sequencing have increasingly been more available and at lower costs. However, no study to date has focused on optimization of viral sample preparation for commercial deep sequencing. Accordingly, the aim of the present study was to evaluate an In-House enzymatic protocol for double-stranded DNA (dsDNA) synthesis and also compare the use of a commercially available kit protocol (Nextera XT, Illumina Inc, San Diego, CA, USA) and its combination with a library quantitation kit (Kapa, Kapa Biosystems, Wilmington, MA, USA) for deep sequencing (Illumina Miseq). Two RNA viruses (canine distemper virus and dengue virus) and one ssDNA virus (porcine circovirus type 2) were tested with the optimized protocols. The tested method for dsDNA synthesis has shown satisfactory results and may be used in laboratory setting, particularly when enzymes are already available. Library preparation combining commercial kits (Nextera XT and Kapa) has yielded more reads and genome coverage, probably due to a lack of small fragment recovering at the normalization step of Nextera XT. In addition, libraries may be diluted or concentrated to provide increase on genome coverage with Kapa quantitation. (C) 2015 Elsevier B.V. All rights reserved. |
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Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R))Double-stranded DNALibrary preparationDeep sequencingMiSeqSequence-independent methods for viral discovery have been widely used for whole genome sequencing of viruses. Different protocols for viral enrichment, library preparation and sequencing have increasingly been more available and at lower costs. However, no study to date has focused on optimization of viral sample preparation for commercial deep sequencing. Accordingly, the aim of the present study was to evaluate an In-House enzymatic protocol for double-stranded DNA (dsDNA) synthesis and also compare the use of a commercially available kit protocol (Nextera XT, Illumina Inc, San Diego, CA, USA) and its combination with a library quantitation kit (Kapa, Kapa Biosystems, Wilmington, MA, USA) for deep sequencing (Illumina Miseq). Two RNA viruses (canine distemper virus and dengue virus) and one ssDNA virus (porcine circovirus type 2) were tested with the optimized protocols. The tested method for dsDNA synthesis has shown satisfactory results and may be used in laboratory setting, particularly when enzymes are already available. Library preparation combining commercial kits (Nextera XT and Kapa) has yielded more reads and genome coverage, probably due to a lack of small fragment recovering at the normalization step of Nextera XT. In addition, libraries may be diluted or concentrated to provide increase on genome coverage with Kapa quantitation. (C) 2015 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UNESP Univ Estadual Paulista, Lab Anim &Human Virol, Dept Microbiol &Immunol, Biosci Inst, BR-18618970 Sao Paulo, BrazilUniv Fed Parana, Dept Vet Med, BR-80035050 Curitiba, Parana, BrazilUniv Illinois, Dept Pathobiol, Urbana, IL 61802 USAUNESP Univ Estadual Paulista, Lab Anim &Human Virol, Dept Microbiol &Immunol, Biosci Inst, BR-18618970 Sao Paulo, BrazilFAPESP: 2011/09424-2FAPESP: 2014/13532-3Elsevier B.V.Universidade Estadual Paulista (Unesp)Universidade Federal do Paraná (UFPR)University of IllinoisUllmann, Leila Sabrina [UNESP]Tozato, Claudia de Camargo [UNESP]Malossi, Camila Dantas [UNESP]Cruz, Tais Fukuta da [UNESP]Cavalcante, Raissa Vasconcelos [UNESP]Kurissio, Jacqueline Kazue [UNESP]Cagnini, Didier Quevedo [UNESP]Rodrigues, Marianna Vaz [UNESP]Biondo, Alexander WelkerAraujo, Joao Pessoa [UNESP]2015-10-21T13:11:25Z2015-10-21T13:11:25Z2015-08-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article60-63http://www.sciencedirect.com/science/article/pii/S0166093415001457Journal Of Virological Methods, v. 220, p. 60-63, 2015.0166-0934http://hdl.handle.net/11449/12860410.1016/j.jviromet.2015.04.009WOS:000356112300012Web of Sciencereponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengJournal Of Virological Methods1.7560,858info:eu-repo/semantics/openAccess2021-10-23T22:03:55Zoai:repositorio.unesp.br:11449/128604Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T17:00:34.086494Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R)) |
title |
Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R)) |
spellingShingle |
Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R)) Ullmann, Leila Sabrina [UNESP] Double-stranded DNA Library preparation Deep sequencing MiSeq |
title_short |
Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R)) |
title_full |
Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R)) |
title_fullStr |
Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R)) |
title_full_unstemmed |
Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R)) |
title_sort |
Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R)) |
author |
Ullmann, Leila Sabrina [UNESP] |
author_facet |
Ullmann, Leila Sabrina [UNESP] Tozato, Claudia de Camargo [UNESP] Malossi, Camila Dantas [UNESP] Cruz, Tais Fukuta da [UNESP] Cavalcante, Raissa Vasconcelos [UNESP] Kurissio, Jacqueline Kazue [UNESP] Cagnini, Didier Quevedo [UNESP] Rodrigues, Marianna Vaz [UNESP] Biondo, Alexander Welker Araujo, Joao Pessoa [UNESP] |
author_role |
author |
author2 |
Tozato, Claudia de Camargo [UNESP] Malossi, Camila Dantas [UNESP] Cruz, Tais Fukuta da [UNESP] Cavalcante, Raissa Vasconcelos [UNESP] Kurissio, Jacqueline Kazue [UNESP] Cagnini, Didier Quevedo [UNESP] Rodrigues, Marianna Vaz [UNESP] Biondo, Alexander Welker Araujo, Joao Pessoa [UNESP] |
author2_role |
author author author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) Universidade Federal do Paraná (UFPR) University of Illinois |
dc.contributor.author.fl_str_mv |
Ullmann, Leila Sabrina [UNESP] Tozato, Claudia de Camargo [UNESP] Malossi, Camila Dantas [UNESP] Cruz, Tais Fukuta da [UNESP] Cavalcante, Raissa Vasconcelos [UNESP] Kurissio, Jacqueline Kazue [UNESP] Cagnini, Didier Quevedo [UNESP] Rodrigues, Marianna Vaz [UNESP] Biondo, Alexander Welker Araujo, Joao Pessoa [UNESP] |
dc.subject.por.fl_str_mv |
Double-stranded DNA Library preparation Deep sequencing MiSeq |
topic |
Double-stranded DNA Library preparation Deep sequencing MiSeq |
description |
Sequence-independent methods for viral discovery have been widely used for whole genome sequencing of viruses. Different protocols for viral enrichment, library preparation and sequencing have increasingly been more available and at lower costs. However, no study to date has focused on optimization of viral sample preparation for commercial deep sequencing. Accordingly, the aim of the present study was to evaluate an In-House enzymatic protocol for double-stranded DNA (dsDNA) synthesis and also compare the use of a commercially available kit protocol (Nextera XT, Illumina Inc, San Diego, CA, USA) and its combination with a library quantitation kit (Kapa, Kapa Biosystems, Wilmington, MA, USA) for deep sequencing (Illumina Miseq). Two RNA viruses (canine distemper virus and dengue virus) and one ssDNA virus (porcine circovirus type 2) were tested with the optimized protocols. The tested method for dsDNA synthesis has shown satisfactory results and may be used in laboratory setting, particularly when enzymes are already available. Library preparation combining commercial kits (Nextera XT and Kapa) has yielded more reads and genome coverage, probably due to a lack of small fragment recovering at the normalization step of Nextera XT. In addition, libraries may be diluted or concentrated to provide increase on genome coverage with Kapa quantitation. (C) 2015 Elsevier B.V. All rights reserved. |
publishDate |
2015 |
dc.date.none.fl_str_mv |
2015-10-21T13:11:25Z 2015-10-21T13:11:25Z 2015-08-01 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://www.sciencedirect.com/science/article/pii/S0166093415001457 Journal Of Virological Methods, v. 220, p. 60-63, 2015. 0166-0934 http://hdl.handle.net/11449/128604 10.1016/j.jviromet.2015.04.009 WOS:000356112300012 |
url |
http://www.sciencedirect.com/science/article/pii/S0166093415001457 http://hdl.handle.net/11449/128604 |
identifier_str_mv |
Journal Of Virological Methods, v. 220, p. 60-63, 2015. 0166-0934 10.1016/j.jviromet.2015.04.009 WOS:000356112300012 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Journal Of Virological Methods 1.756 0,858 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
60-63 |
dc.publisher.none.fl_str_mv |
Elsevier B.V. |
publisher.none.fl_str_mv |
Elsevier B.V. |
dc.source.none.fl_str_mv |
Web of Science reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1808128738681946112 |