CompIgS: A Computational Workflow for comparative analysis of related clonotypes within distinct antibody subclasses
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Individual B cells produce antibodies, also known as immunoglobulins (Ig), that target specific antigens. Collectively, the B cells within an individual generate a diverse Ig-repertoire capable of recognizing a wide range of antigens, which underpins adaptive immunity. On a molecular level, this repertoire is created by somatic recombination of variable (V), diversity (D), and joining (J) gene segments during early B cell development, eventually forming a unique VDJ-sequence that encodes for the antigen-binding region of antibodies. Each individual VDJ-sequence defines a clonotype. The nearly indefinite number of individual antigen-binding regions are joined to a limited number of constant regions, that determine the antibody subclass and hence the effector function, such as complement activation, neutralization, and opsonization, among others. A clonotype may contain antibodies of different subclasses that can have different and even opposing functions. These B cell subclones can introduce hypermutations within their VDJ-sequences to alter the antigen-binding affinities affecting antibody properties and clonal selection. Recent advancements in next-generation sequencing enabled high-depth profiling of antibody repertoires. However, current analysis tools provide limited analysis of related Ig clones present within distinct Ig subclasses of the same sample. To address this, we present an open-source computational tool, designed to identify subclone pairs between two antibody subclasses from the sample. The CompIgS (Comparative Igs Analyzer) workflow processes immunoglobulin variable heavy-chain (VH) repertoire data, utilizing ImMunoGeneTics (IMGT) annotations. It incorporates IMGT/HighV-QUEST and IMGT/StatClonotype outputs to standardize V(D)J representations and identifies shared clonotypes present in two distinct antibody subclasses. This approach allows for comparative analysis of clonal expansion and hypermutation. As a case study, we analyzed related IgE and IgG1 clonotypes from a murine model of food allergy in which IgE promotes the development of allergic symptoms, while IgG1 can inhibit allergy development. CompIgS computed various repertoire metrics including clonotype counts, IgE/IgG1 copy number ratios, somatic hypermutation profiles and estimation of different IgE subpopulations in relation to IgG1.
