High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).
High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).
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DOI：
10.1186/s12864-016-2669-3
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发表时间：
2016-06-14
期刊：
影响因子：
4.4
通讯作者：
Johnson EA
中科院分区：
文献类型：
--
作者：
Preston JL;Royall AE;Randel MA;Sikkink KL;Phillips PC;Johnson EA
多态性基因座存在于种群的整个基因组中，并提供物种适应环境变化所需的原始遗传物质。由于 Illumina 测序等标准方法的错误率很高，很难用下一代测序 (NGS) 追踪人群中罕见单核苷酸多态性 (SNP) 的次要等位基因频率。我们开发了一种湿实验室实验方案和变异识别方法，可识别测序和 PCR 错误，称为配对末端低错误测序 (PELE-Seq)。为了测试 PELE-Seq 方法的特异性和敏感性，我们对含有已知稀有等位基因的对照大肠杆菌 DNA 文库进行了测序，这些等位基因的频率范围为总读数的 0.2–0.4%。 PELE-Seq 比标准文库具有更高的特异性和敏感性。然后，我们使用 PELE-Seq 来表征雷马尼线虫种群在实验室适应前后的稀有等位基因，发现次要和稀有等位基因在实验室适应期间的频率可能会发生较大变化。我们开发了一种罕见等位基因检测方法，可减少测序和 PCR 错误，称为 PELE-Seq。使用对照大肠杆菌群体对 PELE-Seq 进行评估，然后用于将野生 C. remanei 群体与实验室适应群体进行比较。 PELE-Seq 方法非常适合研究各种简化代表性测序方法中稀有等位基因的动态，包括靶向扩增子测序、RAD-Seq、ddRAD 和 GBS。 PELE-Seq 还非常适合线粒体和病毒的全基因组测序以及高通量罕见突变筛选。本文的在线版本 (doi:10.1186/s12864-016-2669-3) 包含补充材料，可供授权用户使用。
Polymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing. We have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2–0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation. We have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens. The online version of this article (doi:10.1186/s12864-016-2669-3) contains supplementary material, which is available to authorized users.