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

Area of Science:

• Virology
• Genomics
• Bioinformatics

Background:

• Emerging and re-emerging viruses pose a significant global health threat.
• Current viral genome sequencing methods are often complex and costly, hindering rapid pathogen identification and outbreak response.
• Untargeted nanopore sequencing offers potential for pathogen detection and genomic surveillance.

Purpose of the Study:

• To develop and validate novel, cost-effective, and efficient random-primed Sequencing Method at the 5' end of RNA Template (SMART)-Seq approaches for RNA virus genome sequencing.
• To assess the performance of these methods using viral isolates, clinical samples, and compare them against established techniques.

Main Methods:

• Development of two random-primed SMART-Seq methods: SMART-9N (sequencing-agnostic) and Rapid SMART-9N (compatible with Oxford Nanopore Technologies rapid adapters).
• Application of these methods to Zika virus isolates, Yellow fever virus plasma samples, and SARS-CoV-2 nasopharyngeal samples.
• Comparison with a gold-standard amplicon-based multiplex PCR method and assessment of limit of detection.

Main Results:

• SMART-9N recovered 10kb of the Zika virus genome in a single nanopore read.
• Rapid SMART-9N achieved full genome coverage for Zika virus, with significant cost and time reductions (10 minutes, up to 45% less cost).
• Both methods demonstrated high sensitivity (limit of detection 6 FFU/mL) and superior genome coverage compared to multiplex PCR for clinical samples, including a 18.5kb read for SARS-CoV-2.

Conclusions:

• SMART-9N and Rapid SMART-9N are sensitive, low-input, and long-read compatible alternatives for RNA virus detection and genome sequencing.
• Rapid SMART-9N significantly improves laboratory workflow efficiency by reducing cost, time, and complexity.
• These methods enhance capabilities for viral surveillance and outbreak preparedness.