Tracking The Interplay Between Circulating And Tumor-Infiltrating T Cells Using Ampliseq-Based Ion Torrent Tcr Beta Immune Repertoire Sequencing.

e14518 Background: TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to future cancer immunotherapy. Here we describe a multiplex PCR-based TCRβ sequencing assay that takes advantage of the exceptionally low base-call error rate and long read capability of the Ion S5 530 chip. The S530 chip provides > 15M reads, allowing for sample sequencing in multiplex. The research assay utilizes AmpliSeq technology and Framework 1 and Constant gene targeting primers to provide comprehensive coverage of TCRβ CDR 1, 2 and 3 from RNA input with a hands-on time of less than 30 minutes. CDR region sequences reveal the antigen specificity of the receptor and may be genetically modified to increase TCR affinity for tumor antigen. Methods: We evaluated assay performance by sequencing TCRβ rearrangements from normal donor peripheral blood leukocyte (PBL) cDNA that had been spiked with 30 reference rearrangements taken from the literature. We then used our assay to evaluate the extent of clonal overlap between matched tumor infiltrating lymphocyte (TIL) and peripheral blood leukocyte repertoires in an individual with squamous cell carcinoma of lung. Results: Results from sequencing of spike-in reference rearrangements indicate that the assay is accurate and sensitive over 5 logs of input template amount while showing minimal amplification bias. Sequencing of matched PBL and TIL repertoires revealed that a subset of the PBL repertoire (8%) consisted of clones also found in TIL. Technical replicates showed high concordance (r > .96) in the frequency of detected clones, indicating that the results were reproducible and samples were sequenced to an appropriate depth. Conclusions: In summary, these data suggest that AmpliSeq-based multiplex PCR and Ion Torrent sequencing provide unbiased, reproducible, scalable, complete, and accurate information for immune repertoire research sequencing applications.