Next Generation Sequencing Unit and Division of Virology, Faculty of Health Sciences, University of the Free State, Bloemfontein 9300, South Africa.
Department of Biotechnology and Food Science, Durban University of Technology, P.O. Box 1334, Durban 4000, South Africa.
J Virol Methods. 2023 Apr;314:114677. doi: 10.1016/j.jviromet.2023.114677. Epub 2023 Jan 16.
Viral metagenomics is increasingly applied in viral detection and virome characterization. Different extraction and enrichment techniques may be adopted, however, reports on their effective influence on viral recovery is often conflicting. Using a three step enrichment steps, the effect of three extraction kits and the influence of DNase treatment with or without rRNA removal for respiratory RNA virus recovery from nasopharyngeal swab samples was evaluated. The viral cocktail containing six different RNA viruses pooled in equal volume were subjected to the different extraction and enrichment methods, sequenced using the Illumina MiSeq, and analysed using Genome Detective. The PureLink® Viral RNA/DNA Mini Kit (PureLink) was highly efficient with better recovery of all the viral agents in the cocktail. The use of rRNA treatment resulted in increased viral recovery with PureLink and QIAamp® Viral RNA Mini kit, while having comparable recovery rate as DNase only with the QIAamp® MinElute Virus Spin Kit. The observed low reads and genome coverage of some of the viruses could be attributed to their low abundance. Depending on sample matrix, extraction choice and enrichment strategy may influence recovery of respiratory RNA virus in metagenomics studies, therefore individual evaluation and adoption may be necessary for a robust result.
病毒宏基因组学在病毒检测和病毒组学特征分析中得到了越来越多的应用。不同的提取和富集技术可能会被采用，但关于它们对病毒回收的有效影响的报告往往存在冲突。本研究采用三步富集步骤，评估了三种提取试剂盒和是否进行 rRNA 去除的 DNase 处理对鼻咽拭子样本中呼吸道 RNA 病毒回收的影响。将包含六种不同 RNA 病毒的病毒混合物以相等体积混合，然后进行不同的提取和富集方法，使用 Illumina MiSeq 进行测序，并使用 Genome Detective 进行分析。PureLink®病毒 RNA/DNA 迷你试剂盒（PureLink）非常高效，能更好地回收混合物中的所有病毒制剂。使用 rRNA 处理可提高 PureLink 和 QIAamp®病毒 RNA 迷你试剂盒的病毒回收率，而与仅使用 DNase 的 QIAamp®MinElute 病毒旋转试剂盒相比，其回收率相当。一些病毒的低读长和基因组覆盖率可能归因于它们的低丰度。根据样本基质、提取选择和富集策略的不同，可能会影响宏基因组学研究中呼吸道 RNA 病毒的回收，因此可能需要对单个样本进行评估和选择，以获得可靠的结果。
