B lymphocytes generate antibodies to neutralize numerous antigens via RAG endonuclease-initiated V(D)J recombination which joins diverse V and D segments into several J segments. High-throughput genome-wide translocation sequencing (HTGTS) was developed to identify genomic fragments that translocate to a distinct “bait” fragment associated with DNA double-stranded breaks (DSBs). With a single bait primer to linearly amplify across bait-prey junctions for “prey” sequence identification, the linear amplification-mediated HTGTS (LAM-HTGTS) could sensitively detect genome-wide DSBs or translocation in an unbiased way independent of prey sequences. Employing the J-segment-associated coding ends cleaved by RAG as bait fragments, we can capture all the Vs and Ds joined to the bait J segments and thus reveal antibody repertoires in B lymphocyte populations. Such LAM-HTGTS adapted repertoire sequencing (HTGTS-Rep-seq) can easily uncover the productive and non-productive V(D)J joins and complementary determining region 3 (CDR3) information that is important for antigen contact, as well as D usage and RAG “off-targets” that are inviable to previous assays. HTGTS-Rep-seq analysis on the Igk repertoires showed that Vks were widely utilized in spite of their transcription orientations convergent or tandem to those of Jks. However, deletion of the four CTCF-binding elements (CBEs) in the Vk-Jk intervening region greatly increased the usage of Vks proximal to Jks. Strikingly, the usage of Vks transcriptionally convergent to Jks was far more elevated than those in the same transcriptional orientation to Jks, which strongly suggested a role of RAG tracking in mediating increased proximal Vk usage in the absence of Vk-Jk intervening CBEs.

Citation Format: Jiazhi Hu, Sherry G. Lin, Zhaoqing Ba, Zhou Du, Yu Zhang, Frederick W. Alt. Revealing antibody repertoires and RAG tracking by linear amplification mediated-high-throughput genome-wide translocation sequencing [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B087.