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

开发基于乳剂的全谱配对链 T 细胞受体测序方法

• 批准号: 10371136
• 负责人: Michael Birnbaum
• 金额: $ 18.84万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-12 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371136
• 关键词: Algorithms; Antigen Receptors; Antigen Targeting; Antigens; Autoimmunity; Bar Codes; Benchmarking; CD4 Positive T Lymphocytes; CD8B1 gene; Cell physiology; Cells; Computing Methodologies; Cytotoxic T-Lymphocytes; DNA; Development; Disease; Emulsions; Equipment; Event; Functional disorder; Genes; Genetic Recombination; Genome; Genomics; Graft Rejection; Health; Human; Hybrids; Immune System Diseases; Immune system; Immunity; Immunology; Individual; Infection; Infection Control; Innate Immune Response; Link; Major Histocompatibility Complex; Malignant Neoplasms; Mediator of activation protein; Methodology; Methods; Modification; Morbidity - disease rate; Oligonucleotides; Peptides; Process; Proteins; Reagent; Resources; Sampling; Series; Specificity; T Chain; T cell receptor repertoire sequencing; T-Cell Development; T-Cell Receptor; T-Lymphocyte; T-cell receptor repertoire; Techniques; Technology; Time; Transcript; V(D)J Recombination; antigen-specific T cells; base; cell transformation; combinatorial; cost; cost estimate; data quality; droplet sequencing; immune function; insight; next generation sequencing; novel; novel strategies; polypeptide; response; single cell analysis

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.

项目摘要 肽-主要组织相容性复合物的T细胞识别是对感染应答的关键决定因素， 癌症和自身免疫虽然最近的技术进步使人们能够更好地 跟踪和分析T细胞受体库，这些方法是极其资源密集型的和/或 有关键的技术限制。在这里，我们提出了一种新的方法，结合基于仿真的分区， 计算序列去卷积以使得能够大规模确定初始T细胞库， 适度的成本（与现有试剂和测序成本相比，估计约为1/3，000细胞） 对于当前技术，约1美元/电池）。我们将首先开发和验证实验修改，以建立 该技术，包括开发低成本DNA条形码珠，形成细胞/珠乳液，以及 TCR转录物的有效转化和捕获。然后，我们将扩展我们以前的计算方法 为了对TCRα/TCRβ序列的库进行去卷积，并在从健康人获得的T细胞上验证我们的方法， 捐助者。此外，我们将扩展我们的方法，包括寡核苷酸标记的pMHC多聚体， TCRα/TCRβ-pMHC配对的库规模追踪。这些技术将共同努力， T细胞库在非常大的规模，一个进步，必要的机械免疫学和 抗原反应性的计算预测。