Rosenfeld Aaron M, Meng Wenzhao, Chen Dora Y, Zhang Bochao, Granot Tomer, Farber Donna L, Hershberg Uri, Luning Prak Eline T
School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, United States.
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
Front Immunol. 2018 Jun 29;9:1472. doi: 10.3389/fimmu.2018.01472. eCollection 2018.
B cell clones expand and contract during adaptive immune responses and can persist or grow uncontrollably in lymphoproliferative disorders. One way to monitor and track B cell clones is to perform large-scale sampling of bulk cell populations, amplifying, and sequencing antibody gene rearrangements by next-generation sequencing (NGS). Here, we describe a series of computational approaches for estimating B cell clone size in NGS immune repertoire profiling data of antibody heavy chain gene rearrangements. We define three different measures of B cell clone size-copy numbers, instances, and unique sequences-and show how these measures can be used to rank clones, analyze their diversity, and study their distribution within and between individuals. We provide a detailed, step-by-step procedure for performing these analyses using two different data sets of spleen samples from human organ donors. In the first data set, 19 independently generated biological replicates from a single individual are analyzed for B cell clone size, diversity and sampling sufficiency for clonal overlap analysis. In the second data set, B cell clones are compared in eight different organ donors. We comment upon frequently encountered pitfalls and offer practical advice with alternative approaches. Overall, we provide a series of pragmatic analytical approaches and show how different clone size measures can be used to study the clonal landscape in bulk B cell immune repertoire profiling data.
B细胞克隆在适应性免疫反应过程中会扩增和收缩，并且在淋巴增殖性疾病中可能持续存在或不受控制地生长。监测和追踪B细胞克隆的一种方法是对大量细胞群体进行大规模采样，通过下一代测序（NGS）扩增并测序抗体基因重排。在这里，我们描述了一系列计算方法，用于在抗体重链基因重排的NGS免疫组库分析数据中估计B细胞克隆大小。我们定义了三种不同的B细胞克隆大小测量方法——拷贝数、实例和独特序列——并展示了如何使用这些测量方法对克隆进行排名、分析其多样性以及研究它们在个体内部和个体之间的分布。我们提供了一个详细的、逐步的程序，用于使用来自人类器官供体的脾脏样本的两个不同数据集进行这些分析。在第一个数据集中，对来自单个个体的19个独立生成的生物学重复样本进行B细胞克隆大小、多样性和用于克隆重叠分析的采样充足性分析。在第二个数据集中，比较了八个不同器官供体的B细胞克隆。我们对常见的陷阱进行了评论，并提供了使用替代方法的实用建议。总体而言，我们提供了一系列实用的分析方法，并展示了如何使用不同的克隆大小测量方法来研究大量B细胞免疫组库分析数据中的克隆格局。
