Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing

Abstract

Antibody specificity and diversity are generated through the enzymatic splicing of genomic gene segments within each B cell. Antibodies are heterodimers of heavy- and light-chains encoded on separate loci. We studied the antibody repertoire from pooled, splenic tissue of unimmunized, adult female C57BL/6J mice, using high-throughput sequencing (HTS) without amplification of antibody transcripts. We recovered over 90,000 heavy-chain and over 135,000 light-chain immunoglobulin sequences. Individual V-, D-, and J-gene segment usage was uniform among the three mouse pools, particularly in highly abundant gene segments, with low frequency V-gene segments not being detected in all pools. Despite the similar usage of individual gene segments, the repertoire of individual B-cell CDR3 amino acid sequences in each mouse pool was highly varied, affirming the combinatorial diversity in the B-cell pool that has been previously demonstrated. There also was some skewing in the V-gene segments that were detected depending on chromosomal location. This study presents a unique, non-primer biased glimpse of the conventionally housed, unimmunized antibody repertoire of the C57BL6/J mouse.