Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States.
Department of Biomedical Engineering, Cockrell School of Engineering, The University of Texas at Austin, Austin, TX, United States.
Front Immunol. 2018 Feb 5;9:33. doi: 10.3389/fimmu.2018.00033. eCollection 2018.
Unique molecular identifiers (MIDs) have been demonstrated to effectively improve immune repertoire sequencing (IR-seq) accuracy, especially to identify somatic hypermutations in antibody repertoire sequencing. However, evaluating the sensitivity to detect rare T cells and the degree of clonal expansion in IR-seq has been difficult due to the lack of knowledge of T cell receptor (TCR) RNA molecule copy number and a generalized approach to estimate T cell clone size from TCR RNA molecule quantification. This limited the application of TCR repertoire sequencing (TCR-seq) in clinical settings, such as detecting minimal residual disease in lymphoid malignancies after treatment, evaluating effectiveness of vaccination and assessing degree of infection. Here, we describe using an MID Clustering-based IR-Seq (MIDCIRS) method to quantitatively study TCR RNA molecule copy number and clonality in T cells. First, we demonstrated the necessity of performing MID sub-clustering to eliminate erroneous sequences. Further, we showed that MIDCIRS enables a sensitive detection of a single cell in as many as one million naïve T cells and an accurate estimation of the degree of T cell clonal expression. The demonstrated accuracy, sensitivity, and wide dynamic range of MIDCIRS TCR-seq provide foundations for future applications in both basic research and clinical settings.
独特分子标识符 (MIDs) 已被证明可有效提高免疫受体库测序 (IR-seq) 的准确性，特别是在鉴定抗体受体库测序中的体细胞高频突变方面。然而，由于缺乏对 T 细胞受体 (TCR) RNA 分子拷贝数的了解，以及从 TCR RNA 分子定量估计 T 细胞克隆大小的通用方法，评估 IR-seq 中稀有 T 细胞的检测灵敏度和克隆扩增程度一直具有挑战性。这限制了 TCR 受体库测序 (TCR-seq) 在临床环境中的应用，例如在治疗后检测淋巴恶性肿瘤中的微小残留病、评估疫苗接种效果和评估感染程度。在这里，我们描述了一种使用基于独特分子标识符聚类的免疫受体库测序 (MIDCIRS) 方法来定量研究 T 细胞中的 TCR RNA 分子拷贝数和克隆性。首先，我们证明了进行 MID 亚聚类以消除错误序列的必要性。此外，我们表明 MIDCIRS 能够敏感地检测多达 100 万个幼稚 T 细胞中的单个细胞，并准确估计 T 细胞克隆表达的程度。MIDCIRS TCR-seq 的准确性、灵敏度和宽动态范围为未来在基础研究和临床环境中的应用奠定了基础。
