title: Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq(®)).
abstract: 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.
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: leila_ullmann@yahoo.com.br.
authors.forename: Leila Sabrina
authors.initials: LS
authors.lastname: Ullmann
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: cctozato@yahoo.com.br.
authors.forename: Claudia
authors.initials: C
authors.lastname: de Camargo Tozato
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: camilamalossi@gmail.com.
authors.forename: Camila Dantas
authors.initials: CD
authors.lastname: Malossi
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: tfcruz@yahoo.com.br.
authors.forename: Tais Fukuta
authors.initials: TF
authors.lastname: da Cruz
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: racavalcante14@gmail.com.
authors.forename: Raíssa Vasconcelos
authors.initials: RV
authors.lastname: Cavalcante
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: jkkurissio@yahoo.com.br.
authors.forename: Jacqueline Kazue
authors.initials: JK
authors.lastname: Kurissio
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: didiercagnini@gmail.com.
authors.forename: Didier Quevedo
authors.initials: DQ
authors.lastname: Cagnini
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: mvazrodrigues@gmail.com.
authors.forename: Marianna Vaz
authors.initials: MV
authors.lastname: Rodrigues
authors.affiliation: Department of Veterinary Medicine, Paraná Federal University, Curitiba 80035-050, Brazil; Department of Pathobiology, University of Illinois, Urbana-Champaign, IL 61802, USA. Electronic address: abiondo@illinois.edu.
authors.forename: Alexander Welker
authors.initials: AW
authors.lastname: Biondo
authors.affiliation: Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil. Electronic address: jpessoa@ibb.unesp.br.
authors.forename: João Pessoa
authors.initials: JP
authors.lastname: Araujo
mesh_terms: D017925:Circovirus / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003716:Dengue Virus / Q000235:genetics*; D004217:Distemper Virus, Canine / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D004247:DNA
keywords: Deep sequencing; Double-stranded DNA; Library preparation; MiSeq