Analysis of Quasispecies of Avain Leukosis Virus Subgroup J Using Sanger and High-throughput Sequencing.
Analysis of Quasispecies of Avain Leukosis Virus Subgroup J Using Sanger and High-throughput Sequencing.
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利用 Sanger 和高通量测序分析 Avain 白血病病毒 J 亚群的准种
DOI：
10.1186/s12985-016-0559-6
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发表时间：
2016-06-27
期刊：
影响因子：
4.8
通讯作者：
Cui Z
中科院分区：
文献类型：
--
作者：
Meng F;Dong X;Hu T;Liu Y;Zhao Y;Lv Y;Chang S;Zhao P;Cui Z
大卫白血病病毒J亚群（ALV-J）是一个由不同但密切相关的基因组组成的复杂混合体，根据达尔文进化原理，它们被命名为准种，并不断发生变化。方法利用MiSeq平台比较常规Sanger测序和深度测序，研究ALV-J准种动态。结果MiSeq测序的准确性和重现性优于Sanger测序。从单位点突变率和两种测序方法区分优势准种的能力来看，常规Sanger测序由于测序样本量少，具有较高的随机性，而深度测序能更准确地反映准种的组成。同时，利用高通量测序数据替换1000次重复的重采样策略，模拟分析了Sanger测序对准种的研究，结果表明，抗体滴度越高，序列熵越大，常规Sanger测序分析难度越大，优势变异体比例越低。综上所述，深度测序更适合于罕见变异的综合检测。本文提出的模拟Sanger测序也将有助于在基于下一代测序数据的不同选择压力下规范准种研究。
BackgroundAvian leukosis viruses subgroup J (ALV-J) exists as a complex mixture of different, but closely related genomes named quasispecies subjected to continuous change according to the Principles of Darwinian evolution.MethodThe present study seeks to compare conventional Sanger sequencing with deep sequencing using MiSeq platform to study quasispecies dynamics of ALV-J.ResultsThe accuracy and reproducibility of MiSeq sequencing was determined better than Sanger sequencing by running each experiment in duplicate. According to the mutational rate of single position and the ability to distinguish dominant quasispecies with two sequencing methods, conventional Sanger sequencing technique displayed high randomness due to few sequencing samples, while deep sequencing could reflect the composition of the quasispecies more accurately. In the mean time, the research of quasispecies via Sanger sequencing was simulated and analyzed with the aid of re-sampling strategy with replacement for 1000 times repeat from high-throughput sequencing data, which indicated that the higher antibody titer, the higher sequence entropy, the harder analyzing with the conventional Sanger sequencing, resulted in lower ratios of dominant variants.ConclusionsIn sum, deep sequencing is better suited for detecting rare variants comprehensively. The simulation of Sanger sequencing that we propose here will also help to standardize quasispecies researching under different selection pressure based on next-generation sequencing data.