Development of optimal wet-lab and bioinformatics protocols for implementation of RAD sequencing for NERC science

生态遗传学的一个常见问题是野生群体中标记的开发和部署。对于非模式生物来说，这可能是一项耗时且昂贵的任务，并且可能成为实现研究目标的严重障碍。最近，限制位点相关DNA测序（RADSeq）作为一种技术出现，具有同时发现、验证和对任何基因组中的大量标记（数千个）进行稳健评分的潜力。使用超高通量测序技术，每周可以筛选数百个个体。有两个问题阻碍了这项技术的广泛应用：在准备RADSeq样本时遇到的困难，以及对产生的数千万到数亿个序列数据点的分析。在这里，我们建议在GenePool基因组学设施（NERC NBAF的合作中心）建立资源和技术，将这种改变游戏规则的技术提供给英国环境和人口遗传学研究。我们将开发最佳实践，简化和可重复的实验室方法，这些方法将提供-从大样本量生成RADSeq文库的稳健方法，-优化分子索引的应用（允许多路复用），-证明使用多种不同的限制性内切酶（采样独立的位点群体）和-稳健的配对端测序系统（扫描每个RAD位点的更长区域以获取snp）。没有经过验证的软件工具来分析RADSeq数据，我们对内部开发的小数据集的探索表明，序列中的错误模式和数据集中站点的差异表示使得数据处理变得非常重要。我们将为RADSeq数据分析构建易于使用的管道，结合最佳实践的质量检查、错误管理和输出，为第三方软件的进一步分析做好准备。这些管道将用于验证秀丽隐杆线虫模型系统中道入口和其他性状的映射，并提供其他基因组中RAD位点的遗传分析。这些工具和规程随后将在内部提供给NERC科学，并通过培训和出版物进行传播。我们还将以成本价向NERC科学提供经过验证的RADSeq适配器集。我们将使用三个测试系统。主要的实验平台将是一组来自秀丽隐杆线虫野生菌株的重组自交系和新构建的杂交，在那里我们将研究RADSeq标记在全测序基因组中精细定位性状的使用。我们还将构建另外两种生物的测试文库，橡树栎和埋甲虫Nicrophorus vespilloides，以在更大的基因组和没有基因组数据的生物体中检测RADSeq。

RAD ABSTRACT A common problem in ecological genetics is the development and deployment of markers in wild populations. This can be a time-consuming and expensive task for non-model organisms, and can be a serious block to achieving research goals. Recently, restriction-site associated DNA sequencing (RADSeq) has emerged as a technology with the potential to simultaneously discover, validate and score robustly a large number of markers (in the thousands) across any genome. Using ultra-high throughput sequencing technologies it is possible to screen hundreds of individuals per week. Two issues hinder wide take-up of this technology: the difficulties encountered in preparing samples for RADSeq, and the analysis of the tens to hundreds of millions of sequence data points generated. Here we propose to establish in the GenePool genomics facility (a collaborating centre in NERC's NBAF) the resources and know-how to deliver this game-changing technique to UK environmental and population genetics research. We will develop best-practice, streamlined and repeatable laboratory methods that will deliver -robust methods for generating RADSeq libraries from large sample sizes, -optimisation of the application of molecular indexing (to permit multiplexing), -proof of the use of multiple different restriction enzymes (sampling independent populations of sites) and -systems for robust paired-end sequencing (to scan longer regions per RAD site for SNPs). There are no validated software tools for analysis of RADSeq data, and our exploration of the small datasets we have developed in house suggest that patterns of error in sequences and differential representation of sites in datasets makes data processing non-trivial. We will build easy-to use pipelines for RADSeq data analysis, incorporating best-practice quality checking, error management and outputs ready for further analyses in third party software. These pipelines will be used to verify the mapping of dauer entry and other traits in the C. elegans model system, and to deliver genetic analysis of RAD sites in the other genomes. These tools and protocols will subsequently be offered in-house to NERC science, and also disseminated through training and publication. We will also make available the validated RADSeq adapter sets at cost to NERC science. We will use three test systems. The major testbed will be a set of recombinant inbred lines derived from, and newly constructed crosses between, wild strains of the nematode Caenorhabditis elegans, where we will investigate the use of RADSeq markers in fine mapping of traits in a fully-sequenced genome. We will also construct test libraries from two other organisms, the oak Quercus robur and the burying beetle Nicrophorus vespilloides, to examine RADSeq in larger genomes, and in organisms with no genome data existing.