Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing

Wellcome Open Research

article

Ingra M. Claro¹  Mariana S. Ramundo³  Thais M. Coletti³  Camila A. M. da Silva³  Ian N. Valenca³  Darlan S. Candido²  Flavia C. S. Sales¹  Erika R. Manuli³  Jaqueline G. de Jesus²  Anderson de Paula³  Alvina Clara Felix³  Pamela dos Santos Andrade³  Mariana C. Pinho³  William M. Souza⁷  Mariene R. Amorim⁸  José Luiz Proenca-Modena⁸  Esper G. Kallas¹  José Eduardo Levi³  Nuno Rodrigues Faria²  Ester C. Sabino³  Nicholas J. Loman⁴  Joshua Quick⁴ 

[1] Faculdade de Medicina da Universidade de São Paulo;MRC Centre for Global Infectious Disease Analysis, J-IDEA, Imperial College London;Instituto de Medicina Tropical, Faculdade de Medicina da Universidade de São Paulo;School of Biosciences, University of Birmingham;Department of Zoology, University of Oxford;Faculdade de Saúde Pública da Universidade de São Paulo;World Reference Center for Emerging Viruses and Arboviruses and Department of Microbiology and Immunology, University of Texas Medical Branch;Laboratory of Emerging Viruses, Department of Genetics, Microbiology, and Immunology, Institute of Biology, University of Campinas;Experimental Medicine Research Cluster, University of Campinas;DASA

关键词: RNA virus;    metagenomic;    nanopore sequencing;    genomic surveillance;    diagnostic;    ZIKV;    YFV;    SARS-CoV-2;   

DOI  :  10.12688/wellcomeopenres.17170.2

学科分类：内科医学

来源: Wellcome

【 摘 要 】

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.

【 授权许可】

CC BY