Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells

Area of Science:

• Virology
• Genomics
• Molecular Diagnostics

Background:

• Enteroviruses are a significant global health concern, necessitating efficient diagnostic methods.
• Current enterovirus identification and genotyping rely on Sanger sequencing, which can be time-consuming and costly.
• There is a need for faster, more economical, and accurate diagnostic platforms for enteroviruses.

Purpose of the Study:

• To evaluate the feasibility of using nanopore sequencing with Flongle flow cells for human enterovirus genotyping.
• To assess the accuracy, speed, and cost-effectiveness of this novel approach compared to Sanger sequencing.
• To determine if Flongle-based nanopore sequencing can reliably identify enterovirus species and genotypes from clinical samples.

Main Methods:

• PCR amplification of the partial VP1 gene from clinical enterovirus samples.
• Multiplexing barcoded PCR amplicons from multiple samples.
• Sequencing on Flongle flow cells and generating nanopore consensus sequences.
• Comparing nanopore consensus sequences with Sanger sequencing data for accuracy and genotyping performance.

Main Results:

• Accurate identification of enterovirus species and genotypes was achieved for all tested clinical samples.
• High sequence identity (>99.7%) was observed between nanopore consensus and Sanger sequences.
• Phylogenetic analysis confirmed the accuracy of genotyping using Flongle-based nanopore sequencing.
• The assay demonstrated reliability even with increased sample multiplexing on a single flow cell.

Conclusions:

• Flongle-based nanopore sequencing is a feasible and accurate platform for enterovirus identification and genotyping.
• This method offers a fast, reproducible, and cost-effective alternative to traditional Sanger sequencing.
• The low cost and rapid turnaround time make Flongle sequencing suitable for clinical diagnostics.