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

• Published in: Journal of virological methods 2015-08, Vol.220, p.60-63
• Main Authors: Ullmann, Leila Sabrina, de Camargo Tozato, Claudia, Malossi, Camila Dantas, da Cruz, Tais Fukuta, Cavalcante, Raíssa Vasconcelos, Kurissio, Jacqueline Kazue, Cagnini, Didier Quevedo, Rodrigues, Marianna Vaz, Biondo, Alexander Welker, Araujo, João Pessoa
• Format: Article
• Language: English
• Subjects: Canine distemper virus; Circovirus - genetics; Deep sequencing; Dengue virus; Dengue Virus - genetics; Distemper Virus, Canine - genetics; DNA - biosynthesis; DNA, Viral - chemistry; DNA, Viral - genetics; DNA, Viral - isolation & purification; Double-stranded DNA; High-Throughput Nucleotide Sequencing - methods; Humans; Library preparation; MiSeq; Porcine circovirus; RNA, Viral - chemistry; RNA, Viral - genetics; RNA, Viral - isolation & purification
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2015.04.009

•Massive sequencing systems may be used for whole viral genome sequencing.•dsDNA may be obtained with RNase H from cDNA and random primer for ssDNA viruses.•Nextera® XT+Kapa may present a valuable alternative for library preparation. 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.