Evaluation of viral heterogeneity using next-generation sequencing, end-point limiting-dilution and mass spectrometry
Evaluation of viral heterogeneity using next-generation sequencing, end-point limiting-dilution and mass spectrometry
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DOI：
10.3233/isb-2012-0453
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发表时间：
2011-01-01
影响因子：
--
通讯作者：
Khudyakov, Y.
中科院分区：
文献类型：
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作者：
Dimitrova, Z.;Campo, D. S.;Khudyakov, Y.
丙型肝炎病毒的序列研究主要集中在含有高变区1(Hvr1)的病毒扩增子上，以获得可以计算出几个群体遗传学参数的序列样本。测序方法的最新进展使分析来自感染患者的前所未有的病毒变异成为可能，并为了解病毒的进化、耐药性和免疫逃逸提供了新的机会。在本文中，我们比较了三种最新的扩增子分析技术：(I)下一代测序；(Ii)使用终点限制-稀释法分离单个序列变体的克隆性测序，随后进行实时荧光定量聚合酶链式反应和测序；以及(Iii)靶序列碱基特异性切割反应的质谱学。这三项技术被用来评估从38名患者(1a和1b亚型)获得的HVR1扩增片段的宿主内多样性和宿主间遗传相关性。在基于序列的数据和基于质谱学的数据之间，对宿主内多样性的评估差异很大。然而，所有三种技术对宿主间变异性的评估在确定病毒株之间的遗传相关性方面都同样准确。这些结果支持了这三种技术在分子流行病学和群体遗传学研究中的应用。质谱学因其高通量、低成本以及与基于序列的方法的可比性而特别有前景。
Hepatitis C Virus sequence studies mainly focus on the viral amplicon containing the Hypervariable region 1 (HVR1) to obtain a sample of sequences from which several population genetics parameters can be calculated. Recent advances in sequencing methods allow for analyzing an unprecedented number of viral variants from infected patients and present a novel opportunity for understanding viral evolution, drug resistance and immune escape. In the present paper, we compared three recent technologies for amplicon analysis: (i) Next-Generation Sequencing; (ii) Clonal sequencing using End-point Limiting-dilution for isolation of individual sequence variants followed by Real-Time PCR and sequencing; and (iii) Mass spectrometry of base-specific cleavage reactions of a target sequence. These three technologies were used to assess intra-host diversity and inter-host genetic relatedness in HVR1 amplicons obtained from 38 patients (subgenotypes 1a and 1b). Assessments of intra-host diversity varied greatly between sequence-based and mass-spectrometry-based data. However, assessments of inter-host variability by all three technologies were equally accurate in identification of genetic relatedness among viral strains. These results support the application of all three technologies for molecular epidemiology and population genetics studies. Mass spectrometry is especially promising given its high throughput, low cost and comparable results with sequence-based methods.