Characterization of B- and T-Cell Immune Repertoires By Anchored Multiplex PCR and Next-Generation Sequencing

The adaptive immune system is involved in various disease conditions including cancer, chronic infection, autoimmune disease and transplant rejection. Adaptive immunity is mediated by B and T lymphocytes, which are activated upon antigen binding to antigen receptors expressed on their surface. Therefore, the spectrum of these antigen receptors, or immune repertoire (IR), provides a means to monitor adaptive immune responses to disease, vaccination and therapeutic interventions. Next-generation sequencing (NGS) of antigen receptor genes is a valuable tool in the study of disease states and responses to various interventions. Traditional amplicon-based NGS assays use opposing primers for targeted amplification of rearranged antigen receptor genes. Thus, large primer panels are required to capture the extensive combinatorial diversity exhibited by the IR. Quantification from such assays requires a complex system of synthetic controls to account for differential amplification efficiency across segment combinations.

Here, we describe an Anchored Multiplex PCR (AMP)-based NGS assay to analyze the IR, employing a minimal set of gene-specific primers in conjunction with molecular barcodes (MBCs) to reduce amplification bias. AMP uses MBCs ligated to cDNA ends and gene-specific primers for amplification, enabling immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Furthermore, this facilitates CDR3 sequence capture from highly fragmented RNA inputs. We validated the quantitative reproducibility and sensitivity of AMP-based B- and T-cell IR assays using high-quality mRNA isolated from peripheral blood leukocytes and highly fragmented RNA isolated from formalin-fixed paraffin-embedded (FFPE) samples.

Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%. Furthermore, our data indicate that clonal diversity in sequencing data is driven by input quantity, total T-cell number, and, to a lesser degree, mRNA quality. Therefore, AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire. This enables sensitive clone tracking and measurement of lymphocyte diversity from fragmented RNA samples.