Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R))
| Autor: | Camila Dantas Malossi, Didier Quevedo Cagnini, Alexander Welker Biondo, Claudia de Camargo Tozato, Raíssa Vasconcelos Cavalcante, João Pessoa Araújo, Taís F. Cruz, Marianna Vaz Rodrigues, Leila Sabrina Ullmann, Jacqueline Kazue Kurissio |
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| Přispěvatelé: | Universidade Estadual Paulista (Unesp), Universidade Federal do Paraná (UFPR), University of Illinois |
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Circovirus
Library preparation Deep sequencing MiSeq Dengue virus medicine.disease_cause Genome Double-stranded DNA chemistry.chemical_compound Virology medicine Humans Distemper Virus Canine Illumina dye sequencing Whole genome sequencing biology High-Throughput Nucleotide Sequencing DNA Dengue Virus biology.organism_classification Porcine circovirus chemistry DNA Viral Nucleic acid RNA Viral |
| Zdroj: | Web of Science Repositório Institucional da UNESP Universidade Estadual Paulista (UNESP) instacron:UNESP |
| Popis: | Made available in DSpace on 2015-10-21T13:11:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-08-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. UNESP Univ Estadual Paulista, Lab Anim &Human Virol, Dept Microbiol &Immunol, Biosci Inst, BR-18618970 Sao Paulo, Brazil Univ Fed Parana, Dept Vet Med, BR-80035050 Curitiba, Parana, Brazil Univ Illinois, Dept Pathobiol, Urbana, IL 61802 USA UNESP Univ Estadual Paulista, Lab Anim &Human Virol, Dept Microbiol &Immunol, Biosci Inst, BR-18618970 Sao Paulo, Brazil FAPESP: 2011/09424-2 FAPESP: 2014/13532-3 |
| Databáze: | OpenAIRE |
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