Development of an emulsion-based method for repertoire-scale paired-chain T cell receptor sequencing

项目摘要 T细胞对多肽-主要组织相容性复合体的识别是感染反应的关键决定因素， 癌症和自身免疫力。虽然最近的技术进步使人们能够更好地 跟踪和分析T细胞受体谱系，这些方法非常耗费资源和/或 有关键的技术限制。在这里，我们提出了一种新的方法，该方法结合了基于乳液的分割和 计算序列去卷积，以实现大规模确定初始T细胞谱系 成本适中(按当前试剂和测序成本计算，估计约为1/3,000个细胞，与 对于目前的技术，约为1美元/细胞)。我们将首先开发和验证试验性修改以建立 这项技术，包括开发低成本的DNA条形码微珠，形成细胞/微珠乳剂，以及 高效转换和捕获TCR转录本。然后我们将扩展我们以前的计算方法 去卷曲TcRα/TcRβ序列池，并在健康人T细胞上验证我们的方法 捐赠者。此外，我们将扩展我们的方法，以包括标记的pMHC多聚体，从而实现 TCRα/TCRβ-pMHC配对的曲目规模跟踪。这些技术结合在一起，将使追踪工作成为可能 极大规模的T细胞谱系，这是机械性免疫学和 抗原反应性的计算预测。

Project Abstract T cell recognition of peptide-Major Histocompatibility Complexes is a key determinant of response to infection, cancer, and autoimmunity. While there have been recent technological advances that have enabled better tracking and analysis of the T cell receptor repertoire, these approaches are extremely resource intensive and/or have key technical limitations. Here, we propose a novel method that combines emulsion-based partitioning and computational sequence deconvolution to enable large-scale determination of the naive T cell repertoire at modest cost (estimated to be ~$1/3,000 cells at current reagent and sequencing costs, as compared to the ~$1/cell for current techniques). We will first develop and validate the experimental modifications to establish this technique, including development of low-cost DNA barcoding beads, formation of cell/bead emulsions, and efficient conversion and capture of TCR transcripts. We will then extend our previous computational approach to deconvolute pools of TCRα/TCRβ sequences, and validate our method on T cells obtained from healthy donors. In addition, we will extend our methodology to include oligonucleotide tagged pMHC multimers, enabling repertoire-scale tracking of TCRα/TCRβ-pMHC pairings. Together, these technologies will enable efforts to track the T cell repertoire at extremely large scale, an advance necessary for both mechanistic immunology and computational prediction of antigen reactivity.