Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Department of Pathology Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA.
Methods Mol Biol. 2022;2453:317-343. doi: 10.1007/978-1-0716-2115-8_18.
In this method we illustrate how to amplify, sequence, and analyze antibody/immunoglobulin (IG) heavy-chain gene rearrangements from genomic DNA that is derived from bulk populations of cells by next-generation sequencing (NGS). We focus on human source material and illustrate how bulk gDNA-based sequencing can be used to examine clonal architecture and networks in different samples that are sequenced from the same individual. Although bulk gDNA-based sequencing can be performed on both IG heavy (IGH) or kappa/lambda light (IGK/IGL) chains, we focus here on IGH gene rearrangements because IG heavy chains are more diverse, tend to harbor higher levels of somatic hypermutations (SHM), and are more reliable for clone identification and tracking. We also provide a procedure, including code, and detailed instructions for processing and annotation of the NGS data. From these data we show how to identify expanded clones, visualize the overall clonal landscape, and track clonal lineages in different samples from the same individual. This method has a broad range of applications, including the identification and monitoring of expanded clones, the analysis of blood and tissue-based clonal networks, and the study of immune responses including clonal evolution.
在本方法中，我们展示了如何通过下一代测序（NGS）从细胞群体的基因组 DNA 中扩增、测序和分析抗体/免疫球蛋白（IG）重链基因重排。我们专注于人类来源的材料，并说明了如何使用基于批量 gDNA 的测序来检查来自同一个体的不同样本中的克隆结构和网络。虽然批量 gDNA 测序可以在 IG 重（IGH）或κ/λ轻（IGK/IGL）链上进行，但我们在这里重点关注 IGH 基因重排，因为 IG 重链更加多样化，往往携带更高水平的体细胞超突变（SHM），并且更可靠用于克隆鉴定和跟踪。我们还提供了一个包含代码的程序，以及用于处理和注释 NGS 数据的详细说明。从这些数据中，我们展示了如何识别扩增的克隆，可视化整体克隆景观，并跟踪来自同一个体的不同样本中的克隆谱系。这种方法具有广泛的应用，包括扩增克隆的鉴定和监测、血液和组织克隆网络的分析以及包括克隆进化在内的免疫反应研究。
