Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing
Ingra Morales Claro, Mariana Severo Ramundo, Thaís de Moura Coletti, Camila Alves Maia da Silva, Ian Nunes Valença, Darlan S. Candido, Flavia Cristina da Silva Sales, Erika R. Manuli, Jaqueline Góes de Jesus, Anderson Vicente de Paula, Alvina Clara Félix, Pâmela dos Santos Andrade, Mariana C. Pinho, William Marciel de Souza, Mariene R. Amorim, José Luiz Proença‐Módena, Esper G. Kallás, José Eduardo Levi, Nuno Rodrigues Faria, Éster Cerdeira Sabino, Nicholas J. Loman, Joshua Quick
Abstract
<ns3:p>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.</ns3:p> <ns3:p>SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular method 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, ‘SMART-9N’, and a version compatible with barcoded PCR primers available from Oxford Nanopore Technologies, ‘Rapid SMART-9N’, for the detection, characterization, and whole-genome sequencing of RNA viruses. 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 6e00 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.</ns3:p> <ns3:p>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.</ns3:p>