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

摘要生态遗传学中的一个常见问题是在野生种群中开发和部署标记。对于非模式生物来说，这可能是一项既耗时又昂贵的任务，并可能成为实现研究目标的严重障碍。最近，限制酶切位点相关DNA测序(RADSeq)已经成为一种潜在的技术，可以同时发现、验证和稳健地对任何基因组中的大量标记(以数千计)进行评分。使用超高通量测序技术，每周可以对数百人进行筛查。两个问题阻碍了这项技术的广泛采用：为RADSeq准备样本时遇到的困难，以及对产生的数千万到数亿个序列数据点的分析。在这里，我们建议在基因库基因组学设施(NERC的NBAF合作中心)中建立资源和技术，以将这一改变游戏规则的技术提供给英国的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-从大样本大小生成RADSeq文库的强大方法，-分子索引应用的优化(允许多路复用)，-多种不同限制酶的使用证明(对独立的位点种群进行采样)，以及-强大的配对末端测序系统(为SNP扫描每个RAD位点更长的区域)。目前还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型数据集的探索表明，序列中的错误模式和数据集中位置的差异表示使数据处理变得非常重要。我们将为RADSeq数据分析建立易于使用的管道，包括最佳实践质量检查、错误管理和准备在第三方软件中进行进一步分析的输出。这些管道将用于验证线虫模型系统中Dauer入口和其他特征的图谱，并提供其他基因组中RAD位点的遗传分析。这些工具和方案随后将在内部提供给NERC Science，并通过培训和出版进行传播。我们还将向NERC Science提供经过验证的RADSeq适配器集。我们将使用三个测试系统。主要的试验台将是一组来自线虫秀丽线虫野生菌株的重组近交系及其之间新构建的杂交，在那里我们将研究RADSeq标记在全序列基因组特征精细定位中的使用。我们还将从另外两种生物--栎树和埋头甲虫--中构建测试文库，以在更大的基因组中以及在没有基因组数据的生物中检测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.