Next-generation Sequencing (NGS) is proving an important tool in increasing understanding of the human immune system, and thereby cancer immunology. αβ-T cells are the primary constituents of human cell-mediated adaptive immunity. The antigen specificity of each αβ-T cell is encoded in the 500-600 bp transcript encompassing the variable portion of the rearranged TCRα and TCRβ subunits, which can be read via NGS in a process termed repertoire sequencing. Until now, the main challenge the field faces is the lack of a technology that can provide a contiguous read of 600 bp to minimize the complexity of designing bias-prone primers and informatics challenges of stitching short reads. Here we leverage the long read capability of Ion 530™ chip to comprehensively sequence all three CDR domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire profiling studies. Initial testing with Leukocyte total RNA demonstrates that this multiplex PCR assay produced repertoires that were much more similar to data derived from 5’RACE protocol than the commonly used BIOMED2 primer set. This result suggested that the use of long reads minimizes bias by allowing targeting of less variable regions. To further assess the performance of the assay, we designed a model system of 30 plasmid controls containing common human T-cell CDR3 sequences. Each plasmid was amplified individually and sequenced to confirm the detection of a single clonal population. Analytical sensitivity of the assay and accuracy of the accompanied analysis solution were further evaluated by spiking in plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results showed the assay offers linearity over 5 orders of magnitude of decreasing input concentration. In summary, we have demonstrated a NGS workflow for TCRβ sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less than 48 hours. For Research Use Only. Not for use in diagnostic procedures.
Citation Format: Denise S. Topacio-Hall, Tim Looney, Yongming Sun, Lauren Miller, Elizabeth Linch, Geoffrey Lowman, Lifeng Lin, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the human TCRβ repertoire on the Ion S5™ System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2017-3567