Qualifying high-throughput immune repertoire sequencing

• Published in: Cellular immunology 2014-03, Vol.288 (1-2), p.31-38
• Main Authors: Niklas, Norbert, Pröll, Johannes, Weinberger, Johannes, Zopf, Agnes, Wiesinger, Karin, Krismer, Konstantin, Bettelheim, Peter, Gabriel, Christian
• Format: Article
• Language: English
• Subjects: Base Sequence; Big data visualization; Case-Control Studies; Clone Cells; Clonotyping; Diversity generation; Genome, Human; Germ-Line Mutation; High-Throughput Nucleotide Sequencing - standards; Humans; Immune repertoire; Immunoglobulin heavy chain; Leukemia, Lymphocytic, Chronic, B-Cell - genetics; Leukemia, Lymphocytic, Chronic, B-Cell - immunology; Leukemia, Lymphocytic, Chronic, B-Cell - pathology; Molecular Sequence Data; Next-generation sequencing; Phylogeny; Receptors, Antigen, B-Cell - classification; Receptors, Antigen, B-Cell - genetics; Receptors, Antigen, B-Cell - immunology; Receptors, Antigen, T-Cell - classification; Receptors, Antigen, T-Cell - genetics; Receptors, Antigen, T-Cell - immunology; Sequence Alignment; Sequence Homology, Nucleic Acid; Somatic hypermutation; T cell receptor β; VDJ recombination
• ISSN: 0008-8749; 1090-2163
• DOI: 10.1016/j.cellimm.2014.02.001

•1,433,471 individual IGHV (B cell) and TCRβ (T cell) VDJ sequences of five healthy control and five CLL samples were compared.•Analysis strategy for diversity assessment of T and B cells.•Targeted resequencing with 454 next-generation sequencing of recombinant genomic DNA after multiplex PCR.•Systemic immunological activity finds its expression in significantly reduced clonality of CLL samples whereas mutation rate is comparable. Diversity of B and T cell receptors, achieved by gene recombination and somatic hypermutation, allows the immune system for recognition and targeted reaction against various threats. Next-generation sequencing for assessment of a cell’s gene composition and variation makes deep analysis of one individual’s immune spectrum feasible. An easy to apply but detailed analysis and visualization strategy is necessary to process all sequences generated. We performed sequencing utilizing the 454 system for CLL and control samples, utilized the IMGT database and applied the presented analysis tools. With the applied protocol, malignant clones are found and characterized, mutational status compared to germline identity is elaborated in detail showing that the CLL mutation status is not as monoclonal as generally thought. On the other hand, this strategy is not solely applicable to the 454 sequencing system but can easily be transferred to any other next-generation sequencing platform.