One step ultra-high-throughput sequencing of the paired antibody VH:VL and TCRβ:α repertoires using cell lysate resistant xenopolymerase in emulsion

Abstract Antibodies and T cell receptors (TCRs) have critical roles in adaptive immunity and thus the determination of their sequences is essential for understanding immune responses in infections or vaccinations, for the discovery of therapeutic antibodies, and for the engineered T cell therapies. Antibodies are composed of variable heavy (VH) and light (VL) chains, and TCRs are composed of variable TCRα/γ and TCRβ/δ chains. Since those chains are encoded by two different transcripts, the determination of functional sequences requires single-cell sequencing. Emulsification of single-cells followed by barcoding of the transcripts or fusion of VH:VL or TCRβ:α transcripts using overlap extension RT-PCR allows high-throughput single-cell sequencing; however, because cell lysate in emulsion inhibits the RT-PCR reaction, these methods require complex microfluidics devices to bypass the inhibition which most of biomedical laboratories cannot access. We discovered recently engineered xenopolymerase, RTX, has an exceptional cell lysate resistance that can overcome this limitation. By employing this unique feature of RTX, we have developed a one-step emulsion-based ultra-high-throughput sequencing method for paired antibody VH:VL and TCRβ:α repertoires not requiring microfluidics or any other specialized equipment. Using this methodology, we obtained high yields (5,000 to >20,000/sample) of paired VH:VL or TCRβ:α clonotypes at low cost. As a demonstration, we applied the methodology to the peripheral blood plasmablasts and T follicular helper (TFH) cells from a seasonal influenza vaccine recipient and discovered high-affinity influenza-specific antibodies and TCRβ:α.