This strategy is based on a technological optimization relying on Rolling Circle Amplification (RCA), combined with a newly designed set of oligonucleotide primers based on a thorough analysis of the IMGT/LIGM-DB database 10. Recently, we have described an original strategy allowing to improve the library construction process and increase its diversity 9. Care needs to be taken to ensure the coverage of a large antibody sequence diversity in order to mimic the natural B cell repertoire as close as possible. Generation of antibody libraries is a crucial step in the attempt to study in vivo immune repertoires 7, 8. Studies have shown that this vast diversity, as well as other characteristics of the immune repertoire, can be influenced by factors such as immunization 2, 3 or pathology, notably autoimmune diseases 4, 5, 6. Finally, somatic hypermutations focused on Complementary Determining Regions (CDR) supplement the mechanisms of immunoglobulin maturation, expanding still further the diversity and leading to affine and specific antibodies. Additionally, at the junctions of V-D and D-J segments, a process of random deletion and addition of nucleotides creates an immense junctional diversity. In the case of B cell receptors, rearrangement of variable (V), diversity (D), and joining (J) gene segments in V-Domain creates a combinatorial diversity for the immunoglobulin heavy chain (IGH), whereas rearrangement of V and J gene segments provides a similar diversity for the lambda or kappa light chains (IGL/IGK) 1 (Fig. This vast diversity of immunoglobulin sequences is not provided by the limited number of genes present in the genome, but by rearrangements of the germline at specific loci. The adaptive immune system is capable of producing antibodies against a large number of immunogens. A comparative study of the distribution of immunoglobulin gene subgroups present in the four libraries has revealed shifts in the B cell repertoire originating from differences in genetic background and immunological status of mice. This library, 2.6 × 10 9 in size, is submitted to high throughput sequencing (Next Generation Sequencing, NGS) in order to analyze the gene subgroups encoding for immunoglobulins. In this study, we base our analysis on a murine scFv library previously described and representing four different immune repertoires: i) healthy and naïve, ii) healthy and immunized, iii) autoimmune prone and naïve, and iv) autoimmune prone and immunized. We performed a study evaluating the diversity harbored by different immune repertoires as a function of their physiopathological status. Nevertheless, the methodological approaches to understand these complex interactions are challenging. The relationship between the immune repertoire and the physiopathological status of individuals is essential to apprehend the genesis and the evolution of numerous pathologies.
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