The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within‐population ITS1 sequence variation in a microparasite infecting Daphnia
The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within‐population ITS1 sequence variation in a microparasite infecting Daphnia
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DOI：
10.1111/1755-0998.12396
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发表时间：
2015-11
影响因子：
7.7
通讯作者：
Enrique González-Tortuero;Enrique González-Tortuero;J. Rusek;A. Petrusek;S. Giessler;Dimitrios P. Lyras;Sonja Grath;F. Castro-Monzón;J. Wolinska
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作者：
Enrique González-Tortuero;Enrique González-Tortuero;J. Rusek;A. Petrusek;S. Giessler;Dimitrios P. Lyras;Sonja Grath;F. Castro-Monzón;J. Wolinska
下一代测序（NGS）平台正在取代传统的分子生物学方案，如克隆和桑格测序。然而，NGS平台的准确性很少被测量时，量化基因型或类群的相对频率在人群中。在这里，我们开发了一种新的生物信息学管道（QRS），它汇集了相似的序列变体，并估计了它们在来自人群或社区的NGS数据集中的频率。我们测试了通过454扩增子测序产生的代表性序列的估计频率是否与通过克隆PCR产物的桑格测序获得的频率显著不同。这是通过分析的高度可变的第一个内部转录间隔区（ITS1）的鱼孢子虫Caullerya mesnili，属水蚤的枝角类的微寄生虫的序列变异。该分析也可作为使用该管道研究群体内变异的案例。此外，还使用了一个公开的Illumina数据集来验证社区级数据的管道。总的来说，C的绝对频率有很好的对应关系。从桑格和454平台获得的mesnili ITS1序列。分子方差分析结果表明，该种群结构与其种群结构密切相关. mesnili在不同的湖泊和不同的年份有所不同，与测序平台无关。我们的研究结果不仅支持扩增子测序数据在群体内结构研究中的有用性，还支持QRS管道在Illumina生成的数据上的成功应用。QRS管道及其文档在GNU公共许可证第3版下可以在http://code.google.com/p/quantification-representative-sequences上免费获得。
Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within‐population variation. Additionally, a public Illumina data set was used to validate the pipeline on community‐level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within‐population structure but also the successful application of the QRS pipeline on Illumina‐generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.