Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells
Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells
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DOI：
10.3390/genes10090659
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发表时间：
2019-09-01
期刊：
影响因子：
3.5
通讯作者：
Ramette, Alban
中科院分区：
文献类型：
--
作者：
Graedel, Carole;Miani, Miguel Angel Terrazos;Ramette, Alban
肠道病毒影响全球数百万人，具有重要的临床意义。肠道病毒鉴定和基因分型的标准方法仍然依赖于短诊断扩增子的桑格测序。在这项研究中，我们评估了使用新的流动池“Flongle”进行纳米孔测序的可行性，以快速，具有成本效益和准确的临床样本中的人类肠道病毒基因分型。从多个患者样本中进行部分VP1基因的PCR扩增，在条形码PCR后将其多重化在一起，并在Flongle流动池上进行多次测序。将从将读数映射到参考数据库获得的纳米孔共有序列与它们的桑格序列对应物进行比较。使用不同年份采集的临床标本，我们能够正确识别所有检测样本的肠道病毒种属和基因型，即使在一个流动池上的条形码样本数量加倍时也是如此。跨测序运行的平均序列同一性> 99.7%。系统发育分析表明，Flongle获得的一致序列提供了准确的基因分型。我们的结论是，新的Flongle为基础的检测，其快速的周转时间，低成本的投资，低成本的每个样品代表了一个准确的，可重复的，具有成本效益的平台，肠道病毒的鉴定和基因分型。
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.