Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing.
Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing.
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使用SMART 9N扩增和纳米测序的快速病毒宏基因组学。
DOI：
10.12688/wellcomeopenres.17170.2
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发表时间：
2021
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新出现和再出现的病毒是一个全球卫生问题。基因组测序作为一种监测流行病毒的方法目前受到复杂和昂贵方法的阻碍。非靶向的宏基因组纳米孔测序可以提供基因组信息，以识别病原体，为暴发做准备，甚至预防暴发。SMART （RNA模板5 '端开关机制）是RNA- seq的常用方法，但目前大多数方法依赖于oligo-dT引物来靶向多腺苷化的mRNA分子。我们开发了两种随机引物SMART-Seq方法，一种测序不可知方法“SMART-9N”和一种版本兼容的快速适配器，可从牛津纳米孔技术公司“快速SMART-9N”获得。这些方法是利用病毒分离物、临床样本开发的，并与金标准的基于扩增子的方法进行比较。从寨卡病毒分离物中，SMART-9N方法在单个纳米孔读取中恢复了10.8kb RNA基因组中的10kb。我们还使用Rapid SMART-9N在高深度覆盖下获得了全基因组覆盖，该方法仅需10分钟，成本比其他方法低45%。结果表明，SMART-9N和Rapid SMART-9N的检出限分别为6个焦点形成单位（focus forming units， FFU）/mL，基因组覆盖率分别为99.02%和87.58%。选择先前经RT-qPCR确认的具有广泛ct值的黄热病病毒血浆样本和SARS-CoV-2鼻咽样本进行验证。与多重PCR方法相比，这两种方法都产生了更大的基因组覆盖率，我们在SARS-CoV-2临床样本中获得了本研究的最长单次读取（18.5 kb），使用Rapid SMART-9N方法获得了60%的病毒基因组。这项工作表明，SMART-9N和Rapid SMART-9N是RNA病毒检测和基因组测序的敏感、低输入和长读取兼容的替代方案，并且Rapid SMART-9N降低了实验室工作的成本、时间和复杂性。
Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach ‘SMART-9N’ and a version compatible rapid adapters available from Oxford Nanopore Technologies ‘Rapid SMART-9N’. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.