用于抗体发现的天然配对 VH:VL 抗体库的生物信息学分析。
Bioinformatic Analysis of Natively Paired VH:VL Antibody Repertoires for Antibody Discovery.
机构信息
The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA.
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS, USA.
出版信息
Methods Mol Biol. 2023;2552:447-463. doi: 10.1007/978-1-0716-2609-2_25.
Next-generation DNA sequencing (NGS) of human antibody repertoires has been extensively implemented to discover novel antibody drugs, to analyze B-cell developmental features, and to investigate antibody responses to infectious diseases and vaccination. Because the antibody repertoire encoded by human B cells is highly diverse, NGS analyses of antibody genes have provided a new window into understanding antibody responses for basic immunology, biopharmaceutical drug discovery, and immunotherapy. However, many antibody discovery protocols analyze the heavy and light chains separately due to the short-read nature of most NGS technologies, whereas paired heavy and light chain data are required for complete antibody characterization. Here, we describe a computational workflow to process millions of paired antibody heavy and light chain DNA sequence reads using the Illumina MiSeq 2x300 NGS platform. In this workflow, we describe raw NGS read processing and initial quality filtering, the annotation and assembly of antibody clonotypes relating to paired heavy and light chain antibody lineages, and the generation of complete heavy+light consensus sequences for the downstream cloning and expression of human antibody proteins.
下一代 DNA 测序（NGS）的人类抗体库已经广泛应用于发现新的抗体药物，分析 B 细胞发育特征，以及研究针对传染病和疫苗的抗体反应。由于人类 B 细胞所编码的抗体库具有高度多样性，因此 NGS 分析抗体基因为理解基础免疫学、生物制药药物发现和免疫疗法中的抗体反应提供了一个新的窗口。然而，由于大多数 NGS 技术的短读长性质，许多抗体发现方案都分别分析重链和轻链，而配对的重链和轻链数据则是完整抗体特征描述所必需的。在这里，我们描述了一种计算工作流程，用于使用 Illumina MiSeq 2x300 NGS 平台处理数百万对的抗体重链和轻链 DNA 序列读段。在这个工作流程中，我们描述了原始 NGS 读段处理和初始质量过滤、与配对重链和轻链抗体谱系相关的抗体克隆型的注释和组装，以及完整的重链+轻链共识序列的生成，用于下游克隆和人类抗体蛋白的表达。
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