Claro Ingra M, Ramundo Mariana S, Coletti Thais M, da Silva Camila A M, Valenca Ian N, Candido Darlan S, Sales Flavia C S, Manuli Erika R, de Jesus Jaqueline G, de Paula Anderson, Felix Alvina Clara, Andrade Pamela Dos Santos, Pinho Mariana C, Souza William M, Amorim Mariene R, Proenca-Modena José Luiz, Kallas Esper G, Levi José Eduardo, Faria Nuno Rodrigues, Sabino Ester C, Loman Nicholas J, Quick Joshua
Faculdade de Medicina da Universidade de São Paulo, Sao Paulo, 05403-000, Brazil.
MRC Centre for Global Infectious Disease Analysis, J-IDEA, Imperial College London, London, SW7 2AZ, UK.
Wellcome Open Res. 2023 Apr 24;6:241. doi: 10.12688/wellcomeopenres.17170.2. eCollection 2021.
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.
新出现和再次出现的病毒是全球卫生关注的问题。基因组测序作为监测流行病毒的一种方法，目前受到复杂且昂贵的方法的阻碍。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、为疫情做准备甚至预防疫情爆发。SMART（RNA模板5'端的切换机制）是RNA测序的一种常用方法，但目前大多数方法依赖于oligo-dT引物来靶向多聚腺苷酸化的mRNA分子。我们开发了两种随机引物SMART-Seq方法，一种与测序无关的方法“SMART-9N”和一种与牛津纳米孔技术公司提供的快速适配器兼容的版本“快速SMART-9N”。这些方法是使用病毒分离株、临床样本开发的，并与基于扩增子的金标准方法进行了比较。从寨卡病毒分离株中，SMART-9N方法在一次纳米孔读取中获得了10.8kb RNA基因组中的10kb。我们还使用快速SMART-9N获得了高深度覆盖的全基因组覆盖，该方法仅需10分钟，成本比其他方法低45%。我们发现这些方法的检测限为6个蚀斑形成单位（FFU）/mL，SMART-9N和快速SMART-9N的基因组覆盖率分别为99.02%和87.58%。选择先前通过具有广泛Ct值的RT-qPCR确认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高的基因组覆盖率，并且我们使用SARS-CoV-2临床样本获得了本研究中最长的单读长（18.5kb），使用快速SMART-9N方法获得了病毒基因组的60%。这项工作表明，SMART-9N和快速SMART-9N是用于RNA病毒检测和基因组测序的灵敏、低输入且与长读长兼容的替代方法，快速SMART-9N改善了实验室工作的成本、时间和复杂性。
