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.next-generation sequencing | B-cell repertoire | adaptive immunity | somatic hypermutation | variable gene segment T he production by B cells of immunoglobulin (Ig) proteins, which are expressed on the cell surface as B-cell receptors and secreted by subsets of B cells as antibodies, is a key component of the adaptive immune system in humans. Through their specific binding to an enormously diverse range of foreign bodies, Ig proteins are able to elicit further immunological response and provide protection. These proteins are assembled in B cells from two pairs of polypeptide chains, termed heavy and light. The antigen-binding portions of these genes are created through the somatic recombination of gene segments, termed variable (V), diversity (D), and joining (J). During the recombination process, one each of the ∼46 V, 23 D, and 6 J gene segments (1) recombine to make the antigen-binding region of the heavy chain; the light chain is created by a similar process, although involving one of two different loci (λ and κ) containing V and J genes only. Over three million different Ig sequences can be created through this V(D)J recombinatorial process alone (2). The potential diversity of these sequences is further expanded to the order of trillions (2) when combined with the random insert...

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