Abstract
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 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. 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.
Highlights
RNA viruses are responsible for causing a broad range of human and veterinary diseases
This sample was used to assess the performance of all three methods: multiplex PCR, SMART-9N, and Rapid SMART-9N, and the metagenomics approaches, SMART-9N, and Rapid SMART-9N, was tested in different serial ten-fold MEM dilutions up to 1–1,000,000 to assess the limit of detection (Extended data: Tables S1 and S222)
Compared to multiplex PCR, the total time of hands-on laboratory work dropped 15% and 61% for the SMART-9N and Rapid SMART-9N respectively, and reagent costs were reduced by 40% and 45% (Figure 1)
Summary
RNA viruses are responsible for causing a broad range of human and veterinary diseases. RNA viruses have been a major cause of emerging and re-emerging infections, including Zika virus (ZIKV), Dengue virus (DENV), Human Immunodeficiency Virus (HIV), Ebola virus (EBOV), yellow fever virus (YFV), and recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic tests can be limited by low specificity, in the case of serological tests, or require a priori knowledge of the viruses to be targeted in the case of RT-PCR (reverse transcription-polymerase chain reaction). For these reasons, acute febrile illness often remains undiagnosed, leading to a failure of epidemiological surveillance. Rapid genomic surveillance systems are essential to identify emerging viruses, detect and monitor viral diversity, and be able to prepare for or even prevent new outbreaks
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