Table_2_Addressing IGHV Gene Structural Diversity Enhances Immunoglobulin Repertoire Analysis: Lessons From Rhesus Macaque.xlsx

The accurate germline gene assignment and assessment of somatic hypermutation in antibodies induced by immunization or infection are important in immunological studies. Here, we illustrate issues specific to the construction of comprehensive immunoglobulin (IG) germline gene reference databases for outbred animal species using rhesus macaques, a frequently used non-human primate model, as a model test case. We demonstrate that the genotypic variation found in macaque germline inference studies is reflected in similar levels of gene diversity in genomic assemblies. We show that the high frequency of IG heavy chain V (IGHV) region structural and gene copy number variation between subjects means that individual animals lack genes that are present in other animals. Therefore, gene databases compiled from a single or too few animals will inevitably result in inaccurate gene assignment and erroneous SHM level assessment for those genes it lacks. We demonstrate this by assigning a test macaque IgG library to the KIMDB, a database compiled of germline IGHV sequences from 27 rhesus macaques, and, alternatively, to the IMGT rhesus macaque database, based on IGHV genes inferred primarily from the genomic sequence of the rheMac10 reference assembly, supplemented with 10 genes from the Mmul_051212 assembly. We found that the use of a gene-restricted database led to overestimations of SHM by up to 5% due to misassignments. The principles described in the current study provide a model for the creation of comprehensive immunoglobulin reference databases from outbred species to ensure accurate gene assignment, lineage tracing and SHM calculations.

在免疫学研究领域，对免疫接种或感染诱导产生的抗体开展精准的生殖系基因归属（germline gene assignment）与体细胞超突变（somatic hypermutation, SHM）水平评估，具有重要学术价值。本研究以当前常用的非人灵长类动物模型——恒河猴作为模式测试对象，阐明了远交群动物的全面免疫球蛋白（immunoglobulin, IG）生殖系基因参考数据库构建过程中面临的专属问题。本研究证实，恒河猴生殖系基因推断研究中观测到的基因型变异，在基因组组装序列的基因多样性水平上得到了印证。研究发现，个体间免疫球蛋白重链V区（immunoglobulin heavy chain V, IGHV）结构与基因拷贝数变异的发生频率较高，这导致部分个体缺失其他个体中存在的相关基因。因此，仅基于单一个体或过少个体构建的基因数据库，必然会对其未涵盖的基因做出不准确的归属判定，进而错误评估该类基因的体细胞超突变水平。本研究通过将一个恒河猴IgG文库分别比对至两类数据库验证了上述结论：一类为KIMDB，即由27只恒河猴的生殖系IGHV序列构建而成的数据库；另一类为IMGT恒河猴数据库，该数据库主要基于rheMac10参考基因组组装序列推断得到的IGHV基因构建，并补充了Mmul_051212组装序列中的10个基因。研究结果显示，由于基因归属判定出现偏差，使用这类受限基因数据库会导致体细胞超突变水平被高估最高达5%。本研究提出的构建原则，可为远交群物种的全面免疫球蛋白参考数据库构建提供参考范式，从而保障生殖系基因归属、谱系追踪以及体细胞超突变水平计算的准确性。