Automated analysis of high-throughput B-cell sequencing data reveals a high frequency of novel immunoglobulin V gene segment alleles
Automated analysis of high-throughput B-cell sequencing data reveals a high frequency of novel immunoglobulin V gene segment alleles
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DOI：
10.1073/pnas.1417683112
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发表时间：
2015-02-24
影响因子：
11.1
通讯作者：
Kleinstein, Steven H.
中科院分区：
文献类型：
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作者：
Gadala-Maria, Daniel;Yaari, Gur;Kleinstein, Steven H.
生殖细胞和表达的B细胞免疫球蛋白（IG）库的个体差异与衰老、疾病易感性以及对感染和疫苗接种的不同反应有关。现在可以通过下一代重排IG基因测序来大规模研究基因库特性。对这些谱系测序（Rep-Seq）数据的准确分析需要识别每个IG序列使用的种系变量（V）、多样性（D）和连接（J）基因片段。目前的V（D）J分配方法通过将序列与已知种系V（D）J区段等位基因的数据库比对来工作。然而，现有的数据库可能是不完整的，新的多态性是很难区分频繁发生的体细胞超突变的IG序列。在这里，我们开发了一个工具IG基因型阐明通过Rep-Seq（TIGGER）。TIGGER分析Rep-Seq数据中的突变模式以鉴定新的V片段等位基因，并且还构建包含受试者携带的特定等位基因组的个性化种系数据库。这些信息将用于改进现有工具（如IMGT/HighV-QUEST）的初始V段分配。将TIGGER应用于来自7名受试者的Rep-Seq数据，确定了11个新的V片段等位基因，包括每个受试者中至少一个。这些新等位基因占这些受试者中独特等位基因总数的13%，并影响3%的V（D）J片段分配。这些结果加强了人类IG V基因的高度多态性，并表明许多新的等位基因仍有待发现。将TIGGER整合到Rep-Seq处理管道中将提高V片段分配的准确性，从而改善B细胞库分析。
Individual variation in germline and expressed B-cell immunoglobulin (Ig) repertoires has been associated with aging, disease susceptibility, and differential response to infection and vaccination. Repertoire properties can now be studied at large-scale through next-generation sequencing of rearranged Ig genes. Accurate analysis of these repertoire-sequencing (Rep-Seq) data requires identifying the germline variable (V), diversity (D), and joining (J) gene segments used by each Ig sequence. Current V(D) J assignment methods work by aligning sequences to a database of known germline V(D) J segment alleles. However, existing databases are likely to be incomplete and novel polymorphisms are hard to differentiate from the frequent occurrence of somatic hypermutations in Ig sequences. Here we develop a Tool for Ig Genotype Elucidation via Rep-Seq (TIgGER). TIgGER analyzes mutation patterns in Rep-Seq data to identify novel V segment alleles, and also constructs a personalized germline database containing the specific set of alleles carried by a subject. This information is then used to improve the initial V segment assignments from existing tools, like IMGT/HighV-QUEST. The application of TIgGER to Rep-Seq data from seven subjects identified 11 novel V segment alleles, including at least one in every subject examined. These novel alleles constituted 13% of the total number of unique alleles in these subjects, and impacted 3% of V(D) J segment assignments. These results reinforce the highly polymorphic nature of human Ig V genes, and suggest that many novel alleles remain to be discovered. The integration of TIgGER into Rep-Seq processing pipelines will increase the accuracy of V segment assignments, thus improving B-cell repertoire analyses.