Antibody repertoire analytics: the development of a visualization toolkit and its application to high-throughput Ig-seq data

表达抗体（Abs）的NGS（称为Ig-seq）越来越多地用于解决基本的免疫学问题，例如：对病原体、疫苗和治疗性Abs的免疫反应如何不同？健康的抗体库和那些与免疫功能障碍相关的抗体库之间有什么区别？典型的Ig-seq数据集由数百万个源自多个样品的部分mRNA Ab序列组成，每个样品来自单个个体和时间点。有一些方法可以分析这些数据的各个方面，包括注释单个Abs及其之间关系的工具（例如www.imgt.org）。然而，支持与Ig-seq数据集互动的工具并不存在。该项目的目的是开发一个可视化分析Ig-seq的工具包，使生物学家能够快速探索他们的数据，产生新的假设并做出新的发现。优先事项将是支持整个抗体库分析，并表征与疫苗设计和治疗性抗体发现相关的基本特性（库多样性，库内和库之间的抗体收敛，以及库进化的动态）。例如，在筛选治疗性Ab之前，使用两种策略来产生Ab多样性：动物免疫和噬菌体展示。在这两种情况下，人们对衡量多样性和引导多样性的正确方法知之甚少。该工具包可以在此发挥重要作用，使人们更好地了解动物库在应对挑战时的进化（最终导致更好的免疫策略），并帮助识别（并最终填补）现有Ab噬菌体库中的空白。该项目的学生将从事两项互补活动：工具开发和免疫学研究。工具包的开发带来了若干挑战。为了促进丰富的分析，该工具包将采用不同的视觉表示模式（例如网络，密度图）与多种相似性度量（例如遗传距离，CDR 3序列同一性），并提供用于比较库的基本操作。后者特别具有挑战性，因为每个样品捕获了一小部分的库多样性，因此共享序列的数量是真实的体内重叠的差估计;样品质量不同，但用于解决这一问题的最佳标准化策略尚不清楚。该工具包还需要处理不同类型的大型库数据集（例如配对与未配对的重链/轻链）和不同格式（例如IMGT与Kabat编号方案）。学生将从博士学位的早期阶段开始使用UCB进行免疫学研究，帮助设计改善噬菌体展示文库构建的实验，并有助于解释免疫动物的实验数据。在伯克贝克大学，学生将分析从现有疫苗设计合作中产生的抗体库，并对公共Ig-seq数据集进行荟萃分析，包括专注于人类免疫模型的跨物种分析。该项目福尔斯完全属于“数据驱动生物学”的BBSRC战略优先事项。拟议的动物抗体库分析旨在帮助科学家对他们在免疫学研究中使用的动物作出知情选择（与“3R”优先事项有关）;将对来自不同年龄健康个体的样本进行荟萃分析（与“整个生命过程中的健康老龄化”有关）。

NGS of expressed antibodies (Abs), known as Ig-seq, is increasingly used to address fundamental immunological questions, such as: How do immune responses to pathogens, vaccines and therapeutic Abs differ? And what are the differences between healthy Ab repertoires and those associated with immune dysfunction? A typical Ig-seq dataset consists of millions of partial mRNA Ab sequences derived from multiple samples, each from a single individual and timepoint. There are methods for analysing aspects of this data, including tools for annotating individual Abs and the relationships between them (e.g. www.imgt.org). However, tools that support interactive engagement with Ig-seq datasets do not exist.The aim of this project is to develop a toolkit for visually analysing Ig-seq, enabling biologists to rapidly explore their data, generate novel hypotheses and make new discoveries. The priority will be to support whole Ab repertoire analytics and to characterize fundamental properties (repertoire diversity, Ab convergence within and between repertoires, and the dynamics of repertoire evolution) relevant to vaccine design and therapeutic Ab discovery. For example, two strategies are used to generate Ab diversity prior to screening for therapeutic Abs: animal immunization, and phage display. In both cases, the right ways to measure diversity, and to elicit it, are poorly understood. Here the toolkit can play a vital role, engendering a better understanding of animal repertoire evolution in response to challenge (ultimately leading to better immunization strategies), and helping to identify (and ultimately fill) gaps in existing Ab phage libraries.The student on this project will engage in two complementary activities: tool development and immunology research. Toolkit development poses several challenges. To facilitate rich analyses, the toolkit will incorporate different modes of visual representations (e.g. networks, density plots) with multiple similarity metrics (e.g. genetic distance, CDR3 sequence identity) and provide essential operations for comparing repertoires. The latter is particularly challenging because each sample captures a small proportion of repertoire diversity, hence the number of shared sequences is a poor estimate of the true, in vivo overlap; and sample quality varies, but the optimal normalization strategy for addressing this is unclear. The toolkit will also need to handle large repertoire datasets of different types (e.g. paired vs. unpaired heavy/light chains) and in different formats (e.g. IMGT vs. Kabat numbering schemes).The student will undertake immunology research with UCB from an early stage of the PhD, helping to design experiments that improve phage display library construction and contributing to the interpretation of experimental data from immunized animals. At Birkbeck the student will analyse Ab repertoires arising from an existing vaccine design collaboration, and undertake meta-analyses of public Ig-seq datasets, including cross-species analyses focusing on models of human immunity.This project falls squarely within the BBSRC strategic priority of "data driven biology". The proposed analyses of animal Ab repertoires are designed to help scientists make informed choices about the animals they use in immunology research (relevant to the "3Rs" priority); and meta-analyses of samples from healthy individuals of various ages will be undertaken (relevant to "healthy ageing across the lifecourse").