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
Laboratory of Animal and Human Virology, Department of Microbiology and Immunology, Biosciences Institute, UNESP - Univ Estadual Paulista, Botucatu, 18618-970 São Paulo, Brazil.
Department of Veterinary Medicine, Paraná Federal University, Curitiba 80035-050, Brazil; Department of Pathobiology, University of Illinois, Urbana-Champaign, IL 61802, USA.
J Virol Methods. 2015 Aug;220:60-3. doi: 10.1016/j.jviromet.2015.04.009. Epub 2015 Apr 19.
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.
不依赖序列的病毒发现方法已广泛用于病毒的全基因组测序。越来越多不同的病毒富集、文库制备和测序方案可供使用且成本更低。然而，迄今为止尚无研究聚焦于用于商业深度测序的病毒样本制备的优化。因此，本研究的目的是评估一种用于双链DNA（dsDNA）合成的内部酶促方案，并比较使用市售试剂盒方案（美国加利福尼亚州圣地亚哥市Illumina公司的Nextera XT）及其与文库定量试剂盒（美国马萨诸塞州威尔明顿市Kapa Biosystems公司的Kapa）组合进行深度测序（Illumina Miseq）的情况。使用优化后的方案对两种RNA病毒（犬瘟热病毒和登革病毒）和一种单链DNA病毒（猪圆环病毒2型）进行了测试。所测试的dsDNA合成方法显示出令人满意的结果，可用于实验室环境，尤其是在已有酶的情况下。结合商业试剂盒（Nextera XT和Kapa）进行文库制备产生了更多的读数和基因组覆盖度，这可能是由于在Nextera XT的标准化步骤中缺乏小片段回收。此外，使用Kapa定量可对文库进行稀释或浓缩以提高基因组覆盖度。
