Sequencing of the canine immunoglobulin repertoire

Profiling the adaptive immune repertoire using next generation sequencing (NGS) has become common in human medicine, showing promise in characterizing clonal expansion of B cell receptors (BCRs) in patients with lymphoid malignancies. In contrast, most work evaluating BCR repertoires in dogs has employed traditional PCR-based approaches analyzing the IGH locus only. The objectives of this study were to: (1) describe a novel NGS protocol to evaluate canine BCRs; (2) develop a bioinformatics pipeline for processing canine BCR sequencing data; and (3) apply these methods to derive insights into BCR repertoires of healthy dogs and dogs undergoing treatment for B-cell lymphoma. We isolated RNA from PBMCs of healthy dogs and dogs newly diagnosed with intermediate-to-large B-cell lymphoma with intent to pursue chemotherapy. Rapid amplification of cDNA ends and NGS were employed to generate cDNA libraries and evaluate BCR repertoires. We developed an analysis pipeline to process, identify, and quantify these repertoires. This work represents the first use of NGS to profile the complete BCR repertoire in healthy dogs and dogs undergoing treatment for intermediate-to-large B-cell lymphoma, a common canine hematopoietic neoplasm that we used as a model for B-cell clonality. Future studies employing these tools may improve disease tracking, provide earlier recognition of relapses, and ultimately offer better-informed and targeted therapeutics.

利用下一代测序（next generation sequencing, NGS）对适应性免疫组库（adaptive immune repertoire）进行谱型分析，目前已在人类医学领域得到广泛应用，在表征淋巴恶性肿瘤患者体内B细胞受体（B cell receptors, BCRs）的克隆扩增方面展现出良好应用前景。与之相对，当前多数评估犬类B细胞受体组库的研究仅采用基于传统聚合酶链式反应（polymerase chain reaction, PCR）的方法，且仅针对免疫球蛋白重链（IGH）基因座开展分析。

本研究的目标如下：（1）建立一套可用于评估犬B细胞受体的新型NGS实验方案；（2）开发用于处理犬B细胞受体测序数据的生物信息学分析流程（bioinformatics pipeline）；（3）运用上述方法解析健康犬以及接受B细胞淋巴瘤治疗的犬的B细胞受体组库特征。

我们从健康犬以及新诊断为中大型B细胞淋巴瘤、拟接受化疗的犬的外周血单个核细胞（peripheral blood mononuclear cell, PBMC）中提取了RNA，采用快速扩增cDNA末端（rapid amplification of cDNA ends, RACE）技术结合NGS构建cDNA文库，并对B细胞受体组库进行评估。本研究开发了一套用于处理、鉴定及定量上述组库的分析流程。

本研究首次利用NGS对健康犬及接受中大型B细胞淋巴瘤治疗的犬的完整B细胞受体组库进行谱型分析；中大型B细胞淋巴瘤是一种常见的犬类造血系统肿瘤（hematopoietic neoplasm），本研究将其作为B细胞克隆性（clonality）的研究模型。

未来运用本研究开发的工具开展相关研究，有望实现更精准的疾病追踪、更早识别肿瘤复发，并最终为临床提供更具针对性的治疗策略。