The study of complex immunological diseases and tumor microenvironments has progressed through recent developments on sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from B cell receptors (BCR) and T cell receptors (TCRs). One major challenge of immune repertoire sequencing is to accurately capture the structural and sequence complexities of antibodies and TCR genes. We have developed a method for accurate sequencing of full length immune gene repertoires of B cells and T cells from both human and mouse samples. RNA was extracted from tissues and peripheral blood mononuclear cells (PBMCs) and used for reverse transcription, during which unique molecular identifiers (UMIs) were added to discretely barcode each mRNA molecule. BCR- and TCR-specific PCR primers were used to enrich full-length BCR and TCR sequences. We have implemented a data analysis pipeline to assemble the full length BCR/TCR transcripts and to collapse PCR copies of each mRNA fragment into a single consensus sequence using UMIs. UMI incorporation enables the absolute quantification of input RNA molecules and accurate ranking of antibody/TCR clone abundance. Furthermore, this method facilitates detection of distinct and shared clones in tissue and blood samples, allowing identification of disease-specific clones to evaluate immunotherapy effects. Our method accurately and sensitively detects target TCR clones down to 0.01%, enabling minimal residual disease (MRD) assessment.
Our immune repertoire sequencing approach allows accurate clonal determination for both BCR and TCR. This technique is applicable for a variety of applications including design of antibody chains for in vitro synthesis, investigation of T cell infiltration of tumor microenvironments, and monitoring of minimal residual disease in cancer patients.