Abstract
Enteroviruses affect millions of people worldwide and are of significant clinical importance. The standard method for enterovirus identification and genotyping still relies on Sanger sequencing of short diagnostic amplicons. In this study, we assessed the feasibility of nanopore sequencing using the new flow cell “Flongle” for fast, cost-effective, and accurate genotyping of human enteroviruses from clinical samples. PCR amplification of partial VP1 gene was performed from multiple patient samples, which were multiplexed together after barcoding PCR and sequenced multiple times on Flongle flow cells. The nanopore consensus sequences obtained from mapping reads to a reference database were compared to their Sanger sequence counterparts. Using clinical specimens sampled over different years, we were able to correctly identify enterovirus species and genotypes for all tested samples, even when doubling the number of barcoded samples on one flow cell. Average sequence identity across sequencing runs was >99.7%. Phylogenetic analysis showed that the consensus sequences achieved with Flongle delivered accurate genotyping. We conclude that the new Flongle-based assay with its fast turnover time, low cost investment, and low cost per sample represents an accurate, reproducible, and cost-effective platform for enterovirus identification and genotyping.
Highlights
Enteroviruses are RNA viruses and among the most common viruses infecting humans.Enterovirus infections are associated with a broad range of clinical symptoms including mild respiratory illness to severe neurological diseases [1]
We assessed the suitability of the Oxford Nanopore Technologies (ONT) flow cell, “Flongle”, for enterovirus genotyping based on multiplexed amplicons produced from samples of clinical origin
RNA extraction, reverse transcription and PCR, library preparation, sequencing time, automatic sequence analyses, and reporting) may be estimated at around 22.5 h for each multiplexed Flongle run (Figure 1). In this proof-of-concept study, we describe the successful application of the new Flongle flow cell for enterovirus genotyping via nanopore sequencing
Summary
Enteroviruses are RNA viruses and among the most common viruses infecting humans. Enterovirus infections are associated with a broad range of clinical symptoms including mild respiratory illness to severe neurological diseases [1]. Their genomes have high variability due to frequent mutations and recombinations and more than 100 genotypes are known to infect humans [2,3]. Molecular diagnostic tests for genotyping of enteroviruses usually target the VP1 region, which is known to correlate with viral serotype [4], by Sanger sequencing the PCR amplified gene. Sanger sequencing is still the standard for enterovirus amplicon sequencing as generation sequencing (NGS) approaches tend to have longer turnaround times and generally require larger capital investments [5]
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