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

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 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.

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

Figure 1.. Comparison between the steps and cost of the workflows – tiling amplicon sequencing – Multiplex PCR, SMART-9N, and Rapid SMART-9N.

Abbreviations: SSP: Strand Switching Primer. US$, American dollar.

Figure 2.. Comparison of multiplex PCR, SMART-9N, and Rapid SMART-9N approaches.

A) Genome coverage of ZIKV genome for reference material as to coverage of reads mapped to the genome reference position comparing the multiplex PCR, SMART-9N, and Rapid SMART-9N approaches. B) Limit of detection of the SMART-9N and Rapid SMART-9N methods analyzing the proportions of reads mapping to the appropriate reference viral sequence across a range of sample input (FFU/mL) on the left plot and percentage of the reference genome sequenced to a minimum depth of 20-fold in the data generated across a range of sample input (FFU/mL) on the right plot.

Figure 3.. Comparison of multiplex PCR, SMART-9N, and Rapid SMART-9N results for YFV clinical samples.

A) Average genome coverage depth and 95% of reads mapped to the genome reference position. B) Proportion of reads mapping to the reference genome across a range of Ct -values (left) and percentage of the reference genome sequenced to a minimum depth of 20-fold across a range of Ct -values (right). C) N50 of each sample in bp. (n=7 samples).

Figure 4.. Comparison of multiplex PCR, and Rapid SMART-9N results for SARS-CoV-2 clinical samples.

A) Average genome coverage depth and 95% of reads mapped to the genome reference position. B) Proportion of reads mapping to the reference genome across a range of Ct -values (left) and percentage of the reference genome sequenced to a minimum depth of 20-fold across a range of Ct -values (right). C) N50 of each sample in bp. (n=10 samples).