title: Automated analysis of high-throughput B-cell sequencing data reveals a high frequency of novel immunoglobulin V gene segment alleles.
abstract: 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.
authors: Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06511;
authors: Daniel
authors: D
authors: Gadala-Maria
authors: Department of Pathology and Bioengineering Program, Faculty of Engineering, Bar-Ilan University, Ramat Gan 5290002, Israel.
authors: Gur
authors: G
authors: Yaari
authors: Department of Pathology and.
authors: Mohamed
authors: M
authors: Uduman
authors: Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06511; Department of Pathology and Department of Immunobiology, Yale University School of Medicine, Yale University, New Haven, CT 06511; and steven.kleinstein@yale.edu.
authors: Steven H
authors: SH
authors: Kleinstein
mesh_terms: D000483:Alleles; D001331:Automation; D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D030541:Databases, Genetic; D015322:Gene Rearrangement, B-Lymphocyte; D005803:Genes, Immunoglobulin; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D059645:Mutation Rate; D011110:Polymorphism, Genetic; D012984:Software; D060152:V(D)J Recombination / Q000235:genetics
chemical_list: D007135:Immunoglobulin Variable Region
keywords: B-cell repertoire; adaptive immunity; next-generation sequencing; somatic hypermutation; variable gene segment