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

开发最佳湿实验室和生物信息学协议，以实施 NERC 科学的 RAD 测序

• 批准号: NE/H019804/1
• 负责人: Mark Blaxter
• 金额: $ 36.06万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NE%2FH019804%2F1
• 关键词: Development; optimal; wet; lab; bioinformatics

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.

RAD 摘要 生态遗传学中的一个常见问题是野生种群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，并且可能严重阻碍实现研究目标。最近，限制性位点相关 DNA 测序 (RADSeq) 已成为一项技术，能够同时发现、验证和评分任何基因组中的大量标记（数千个）。使用超高通量测序技术，每周可以筛查数百人。有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，以及对生成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）中建立资源和专业知识，将这种改变游戏规则的技术应用于英国环境和群体遗传学研究。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， - 分子索引应用的优化（以允许多重分析）， - 多种不同限制性酶的使用证明（对位点的独立群体进行采样）和 - 稳健的双端测序系统（扫描每个 RAD 位点的较长区域 SNP）。目前还没有经过验证的软件工具可用于分析 RADSeq 数据，而且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，结合最佳实践质量检查、错误管理和输出，为第三方软件中的进一步分析做好准备。这些管道将用于验证线虫模型系统中 dauer 条目和其他性状的映射，并对其他基因组中的 RAD 位点进行遗传分析。这些工具和协议随后将提供给 NERC 科学内部，并通过培训和出版物进行传播。我们还将以 NERC 科学成本提供经过验证的 RADSeq 适配器集。我们将使用三个测试系统。主要的测试平台将是一组源自秀丽隐杆线虫野生品系的重组自交系以及新构建的杂交品系，我们将在其中研究 RADSeq 标记在全测序基因组性状精细定位中的用途。我们还将利用另外两种生物（橡树 Quercus robur 和埋藏甲虫 Nicrophorus vespilloides）构建测试文库，以检查较大基因组和不存在基因组数据的生物体中的 RADSeq。

项目成果

Characterisation of QTL-linked and genome-wide restriction site-associated DNA (RAD) markers in farmed Atlantic salmon.

• DOI: 10.1186/1471-2164-13-244
• 发表时间: 2012-06-15
• 期刊: BMC genomics
• 影响因子: 4.4
• 作者: Houston RD;Davey JW;Bishop SC;Lowe NR;Mota-Velasco JC;Hamilton A;Guy DR;Tinch AE;Thomson ML;Blaxter ML;Gharbi K;Bron JE;Taggart JB
• 通讯作者: Taggart JB

Special features of RAD Sequencing data: implications for genotyping.

• DOI: 10.1111/mec.12084
• 发表时间: 2013-06
• 期刊: Molecular ecology
• 影响因子: 4.9
• 作者: Davey JW;Cezard T;Fuentes-Utrilla P;Eland C;Gharbi K;Blaxter ML
• 通讯作者: Blaxter ML