Using high-throughput sequencing to leverage surveillance of genetic diversity and oseltamivir resistance: a pilot study during the 2009 influenza A(H1N1) pandemic.
Using high-throughput sequencing to leverage surveillance of genetic diversity and oseltamivir resistance: a pilot study during the 2009 influenza A(H1N1) pandemic.
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DOI：
10.1371/journal.pone.0067010
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Martínez-Barnetche J
中科院分区：
文献类型：
--
作者：
Téllez-Sosa J;Rodríguez MH;Gómez-Barreto RE;Valdovinos-Torres H;Hidalgo AC;Cruz-Hervert P;Luna RS;Carrillo-Valenzo E;Ramos C;García-García L;Martínez-Barnetche J
流感病毒表现出高突变率和复杂的进化模式。新一代测序（NGS）已广泛应用于复杂生物样品遗传多样性的定性和半定量评估。由于当前NGS平台的巨大通量，“深度测序”方法可以在目标遗传区域识别罕见的遗传病毒变异，但通常仅限于少量样本。我们设计了一项原理验证研究，以测试将测序通量从高深度-小样本量的方法重新分配到低深度-大样本量的方法是否可行，并有助于流感流行病学监测。利用454-Roche测序技术，我们从2009年5月至11月墨西哥流感大流行期间采集的感染患者（n = 299）鼻拭子样本的48个cDNA条形码文库中，对甲型H1N1流感大流行（a (H1N1)pdm）病毒神经氨酸酶基因的307 bp扩增子进行了较低深度的测序。该方法显示，在大流行第一波（5 - 7月）到第二波（9 - 11月）的过渡期间，NA基因的N248D突变取代了最初的遗传变异，从而能够建立与遗传多样性的时间和地理关联，并鉴定与奥斯他韦耐药性相关的突变。在低测序深度下对NA基因的短扩增子进行NGS测序，可以对大量样本进行遗传筛选，从而深入了解病毒遗传多样性动态和鉴定与奥司他韦耐药性相关的遗传变异。需要进一步的研究来解释在第二次浪潮中观察到的遗传变异的替换。随着测序通量的提高以及文库复用和自动化程度的提高，我们预计本文提出的方法可以扩大到流感和其他传染病的全球遗传监测。
Influenza viruses display a high mutation rate and complex evolutionary patterns. Next-generation sequencing (NGS) has been widely used for qualitative and semi-quantitative assessment of genetic diversity in complex biological samples. The “deep sequencing” approach, enabled by the enormous throughput of current NGS platforms, allows the identification of rare genetic viral variants in targeted genetic regions, but is usually limited to a small number of samples. We designed a proof-of-principle study to test whether redistributing sequencing throughput from a high depth-small sample number towards a low depth-large sample number approach is feasible and contributes to influenza epidemiological surveillance. Using 454-Roche sequencing, we sequenced at a rather low depth, a 307 bp amplicon of the neuraminidase gene of the Influenza A(H1N1) pandemic (A(H1N1)pdm) virus from cDNA amplicons pooled in 48 barcoded libraries obtained from nasal swab samples of infected patients (n = 299) taken from May to November, 2009 pandemic period in Mexico. This approach revealed that during the transition from the first (May-July) to second wave (September-November) of the pandemic, the initial genetic variants were replaced by the N248D mutation in the NA gene, and enabled the establishment of temporal and geographic associations with genetic diversity and the identification of mutations associated with oseltamivir resistance. NGS sequencing of a short amplicon from the NA gene at low sequencing depth allowed genetic screening of a large number of samples, providing insights to viral genetic diversity dynamics and the identification of genetic variants associated with oseltamivir resistance. Further research is needed to explain the observed replacement of the genetic variants seen during the second wave. As sequencing throughput rises and library multiplexing and automation improves, we foresee that the approach presented here can be scaled up for global genetic surveillance of influenza and other infectious diseases.
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作者：
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