Ultra-deep pyrosequencing (UDPS) data treatment to study amplicon HCV minor variants.
Ultra-deep pyrosequencing (UDPS) data treatment to study amplicon HCV minor variants.
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DOI：
10.1371/journal.pone.0083361
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Quer J
中科院分区：
文献类型：
--
作者：
Gregori J;Esteban JI;Cubero M;Garcia-Cehic D;Perales C;Casillas R;Alvarez-Tejado M;Rodríguez-Frías F;Guardia J;Domingo E;Quer J
我们研究了超深度焦磷酸测序（UDPS）方法定量HCV病毒准种的可靠性和重现性。我们的研究分为两部分。首先，通过克隆混合物样品的UDPS测序，我们已经建立了UDPS的全局噪声水平，并微调了先前针对HBV序列分析优化的数据处理工作流程。其次，我们研究了该方法的重现性，在应用克隆/对照研究开发的误差过滤器后，在三个大规模测序平台（FLX+、FLX和Junior）上比较了来自两个患者样本的5个扩增子。在对UDPS结果进行噪声过滤后，三个重复均显示出大于0.7%的12个相同的多态性位点，平均CV为4.86%。两个重复和一个重复分别鉴定出两个低于0.6%的多态位点。GS-Junior、GS-FLX和GS-FLX+分别检测到25、23和26种单倍型。标准化Shannon熵（Sn）、突变频率（Mf）和核苷酸多样性（Pi）的变异系数分别为1.46%、3.96%和3.78%。在GS-FLX和GS-FLX+中，两名患者（各5个扩增子）的Sn、Mf和Pi的平均绝对差异分别为1.46%、3.96%和3.78%。在0.5%以上没有观察到假多态性位点。我们的研究结果表明，UDPS是一个最佳的替代分子克隆定量研究HCV病毒准种群体，无论是在复杂性和组成。我们提出了一种用于来自RNA病毒准种的扩增子的UDPS数据处理工作流程，其在每条链至少10，000个读数的测序深度下，能够获得高于0.5%丰度的共有单倍型的序列和频率，没有错误突变，具有高置信度，抗性突变体作为1%水平的微小变体，具有变体不被遗漏的高置信度，和准物种复杂性的高度可信的测量。
We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.
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