Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R))

Author(s)

Ullmann, Leila Sabrina ^[1] ; Tozato, Claudia de Camargo ^[1] ; Malossi, Camila Dantas ^[1] ; da Cruz, Tais Fukuta ^[1] ; Cavalcante, Raissa Vasconcelos ^[1] ; Kurissio, Jacqueline Kazue ^[1] ; Cagnini, Didier Quevedo ^[1] ; Rodrigues, Marianna Vaz ^[1] ; Biondo, Alexander Welker ^{[2, 3]} ; Araujo, Jr., Joao Pessoa ^[1]

Affiliation

^[1] UNESP Univ Estadual Paulista, Lab Anim & Human Virol, Dept Microbiol & Immunol, Biosci Inst, BR-18618970 Sao Paulo - Brazil

^[2] Univ Fed Parana, Dept Vet Med, BR-80035050 Curitiba, Parana - Brazil

^[3] Univ Illinois, Dept Pathobiol, Urbana, IL 61802 - USA

• Document type: Journal article
• Source: Journal of Virological Methods; v. 220, p. 60-63, AUG 2015.
• Web of Science Citations: 9

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. (C) 2015 Elsevier B.V. All rights reserved. (AU)

• FAPESP's process: 11/09424-2 - Virus research in non-human primates by molecular techniques and metagenomics
• Grantee: Leila Sabrina Ullmann
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 14/13532-3 - Genomic and transcriptomic studies of viral pathogens in animals
• Grantee: João Pessoa Araújo Junior
• Support Opportunities: Regular Research Grants