Characterization of B- and T-cell immune repertoires using Anchored Multiplex PCR and Next-Generation Sequencing.

Abstract

Abstract Introduction NGS-based analysis of the immune repertoire (IR) is a powerful tool to monitor disease, adaptive immune responses to disease, vaccination and therapeutic interventions. IR characterization by NGS usually requires large primer panels, due to the extensive combinatorial diversity, and a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Anchored Multiplex PCR (AMP™) uses molecular barcoded (MBC) adapters and gene-specific primers (GSPs), enabling NGS-based 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. Here, we describe AMP-based NGS assays for IR characterization, Immunoverse™ IGH and TCR, which utilize a minimal set of unidirectional GSPs and MBC adapters that reduce amplification bias. Methods The quantitative reproducibility and sensitivity of our assays was validated using mRNA isolated from PBMCs of healthy donors, B-cell chronic lymphocytic leukemia donors and formalin-fixed paraffin-embedded (FFPE) tissue. Results Both assays demonstrated high reproducibility between replicates with quantitative clone tracking down to 0.01%. The ability to determine isotype, clonotype and IGHV mutational status in a single assay was demonstrated. Preliminary TCR assay data indicates that CDR3 sequence capture is possible from FFPE tissue with clonotype calling being driven by input quantity, T-cell content, and, to a lesser degree, mRNA quality. Conclusions AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.