Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells
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...
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doaj-02aa4848c0c24c40b3480bebf0ae7d292020-11-24T20:42:49ZengMDPI AGGenes2073-44252019-08-0110965910.3390/genes10090659genes10090659Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow CellsCarole Grädel0Miguel Angel Terrazos Miani1Maria Teresa Barbani2Stephen L Leib3Franziska Suter-Riniker4Alban Ramette5Institute for Infectious Diseases, University of Bern, CH-3012 Bern, SwitzerlandInstitute for Infectious Diseases, University of Bern, CH-3012 Bern, SwitzerlandInstitute for Infectious Diseases, University of Bern, CH-3012 Bern, SwitzerlandInstitute for Infectious Diseases, University of Bern, CH-3012 Bern, SwitzerlandInstitute for Infectious Diseases, University of Bern, CH-3012 Bern, SwitzerlandInstitute for Infectious Diseases, University of Bern, CH-3012 Bern, SwitzerlandEnteroviruses 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 <i>VP1</i> 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.https://www.mdpi.com/2073-4425/10/9/659enterovirusNGSnanopore sequencingdiagnostics |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Carole Grädel Miguel Angel Terrazos Miani Maria Teresa Barbani Stephen L Leib Franziska Suter-Riniker Alban Ramette |
spellingShingle |
Carole Grädel Miguel Angel Terrazos Miani Maria Teresa Barbani Stephen L Leib Franziska Suter-Riniker Alban Ramette Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells Genes enterovirus NGS nanopore sequencing diagnostics |
author_facet |
Carole Grädel Miguel Angel Terrazos Miani Maria Teresa Barbani Stephen L Leib Franziska Suter-Riniker Alban Ramette |
author_sort |
Carole Grädel |
title |
Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells |
title_short |
Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells |
title_full |
Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells |
title_fullStr |
Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells |
title_full_unstemmed |
Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells |
title_sort |
rapid and cost-efficient enterovirus genotyping from clinical samples using flongle flow cells |
publisher |
MDPI AG |
series |
Genes |
issn |
2073-4425 |
publishDate |
2019-08-01 |
description |
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 <i>VP1</i> 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. |
topic |
enterovirus NGS nanopore sequencing diagnostics |
url |
https://www.mdpi.com/2073-4425/10/9/659 |
work_keys_str_mv |
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