DataSheet_5_Unraveling the chicken T cell repertoire with enhanced genome annotation.csv

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）谱系测序已成为一种强大的工具，用以解析宿主免疫系统内T细胞的多样性和功能。然而，由于鸡的TCR位点的基因组注释不完整，尽管鸡在农业和免疫学模型中具有重要地位，其TCR谱系仍然理解不足。本研究中，我们通过采用5'快速扩增互补DNA末端（5'RACE）TCR谱系测序以及互补DNA（cDNA）分子的分子条形码技术，解决了这一关键问题。同时，我们增强了鸡基因组中TCR可变（V）、多样性（D，仅存在于β和δ位点）和连接（J）基因的基因组注释。为提高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，我们实现了对所有已知鸡TCR位点的全面注释，包括染色体27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，我们对脾脏的α/β/γ/δTCR谱系进行了表征。对谱系的分析揭示了所有链中均偏好使用特定的V和J组合，而整体特征则表现出无偏见的谱系特点。我们在α和γ链TCR谱系中观察到个体鸟之间中等程度的共享互补决定区3（CDR3）克隆型，包括最频繁出现的克隆型。然而，β和δ谱系在每只鸟中均以独特性为主。总之，我们的TCR谱系分析使我们得以解析鸡中T细胞的组成、多样性和功能。本研究不仅代表了理解鸟类T细胞生物学的一个重要步骤，而且将为宿主-病原体相互作用、疫苗开发和鸟类免疫学的进化历史提供新的见解。