Monitoring the T cell receptor (TCR) repertoire in health and disease can provide key insights into adaptive immune responses, but the accuracy of current TCR sequencing (TCRseq) methods is unclear. In this study, we systematically compared the results of nine commercial and academic TCRseq methods, including six rapid amplification of complementary DNA ends (RACE)-polymerase chain reaction (PCR) and three multiplex-PCR approaches, when applied to the same T cell sample. We found marked differences in accuracy and intra- and inter-method reproducibility for T cell receptor α (TRA) and T cell receptor β (TRB) TCR chains. Most methods showed a lower ability to capture TRA than TRB diversity. Low RNA input generated non-representative repertoires. Results from the 5′ RACE-PCR methods were consistent among themselves but differed from the RNA-based multiplex-PCR results. Using an in silico meta-repertoire generated from 108 replicates, we found that one genomic DNA-based method and two non-unique molecular identifier (UMI) RNA-based methods were more sensitive than UMI methods in detecting rare clonotypes, despite the better clonotype quantification accuracy of the latter.

监测健康和疾病中的 T 细胞受体 (TCR) 库可以为适应性免疫反应提供重要见解，但当前 TCR 测序 (TCRseq) 方法的准确性尚不清楚。在这项研究中，我们系统地比较了九种商业和学术 TCRseq 方法的结果，包括六种互补 DNA 末端快速扩增 (RACE)-聚合酶链式反应 (PCR) 和三种多重 PCR 方法，应用于相同的 T 细胞样本。我们发现 T 细胞受体 α (TRA) 和 T 细胞受体 β (TRB) TCR 链的准确性以及方法内和方法间重现性存在显着差异。大多数方法捕获 TRA 多样性的能力低于捕获 TRB 多样性的能力。低 RNA 输入产生非代表性的库。 5' RACE-PCR 方法的结果彼此一致，但与基于 RNA 的多重 PCR 结果不同。使用由 108 个重复生成的计算机元库，我们发现一种基于基因组 DNA 的方法和两种基于非唯一分子标识符 (UMI) RNA 的方法在检测稀有克隆型方面比 UMI 方法更敏感，尽管克隆型更好后者的量化精度。