NGS-based analysis of the mouse B-cell receptor repertoire

Abstract

The B cell receptor (BCR), especially the complementarity-determining region 3 (CDR3) encoded on the variable region of the immunoglobulin heavy chain, is the most important region that determines antigen binding and specificity. Furthermore, sequence analysis of the CDR3 region is important for understanding the clonal distribution and dynamics in the clonal evolution of leukemic cells. Here we present a sensitive method for fast and precise analysis of the BCR repertoire of mouse chronic lymphocytic leukemia cells by amplification and high-throughput DNA sequencing of the genomic rearranged VDJ locus. Tagging and indexing of the PCR amplified VDJ locus enables multiplexing and Illumina sequencing of up to 3×96 samples (depending on the requested coverage) in parallel on a MiSeq system, with a total coverage of 25 million reads for a high output run. The protocol includes a suggestion for fast bioinformatics analysis using the MiXCR software.