DataSheet_7_Unraveling the chicken T cell repertoire with enhanced genome annotation.zip

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5’ rapid amplification of complementary DNA ends (5’RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in β and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/β/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the β and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.

T 细胞受体（TCR）库测序已成为理解宿主免疫系统内 T 细胞多样性和功能的有力工具。然而，由于 TCR 位点的基因组注释不完整，尽管鸡在农业及免疫学模型中具有重要地位，其鸡 TCR 库仍鲜为人知。在本研究中，我们通过采用 5’ 互补 DNA末端快速扩增（5’RACE）TCR 库测序和互补 DNA（cDNA）分子的分子条形码，解决了这一关键问题。同时，我们提升了鸡基因组中 TCR 可变（V）、多样性（D，仅存在于 β 和 δ 位点）及连接（J）基因的基因组注释。为了提高 TCR 注释的效率，我们开发了 VJ-gene-finder 算法，该算法旨在从脱氧核糖核酸（DNA）序列中提取 VJ 基因候选者。利用此工具，我们实现了所有已知鸡 TCR 位点的全面注释，包括第 27 号染色体上的 α/δ 位点。进化分析揭示了每个位点通过长同源单元的复制而独立进化。为了定义健康鸡的 TCR 多样性基线并展示该方法的可行性，我们表征了脾脏的 α/β/γ/δ TCR 库。对这些库的分析揭示了所有链中特定 V 和 J 组合的偏好性使用，而整体特征则体现了无偏见的库。我们在 α 和 γ 链库中观察到个体鸟类之间共享的互补决定区域 3（CDR3）克隆型别处于中等水平，包括最常出现的克隆型别。然而，β 和 δ 库则主要对每只鸡都是独特的。总之，我们的 TCR 库分析使我们得以揭示鸡中 T 细胞的组成、多样性和功能。这项工作不仅代表了理解鸟类 T 细胞生物学的重大进展，而且将有助于阐明宿主-病原体相互作用、疫苗开发以及鸟类免疫学的进化历史。