Wei Yu-Chun, Pospiech Mateusz, Meng Yiting, Alachkar Houda
Department of Clinical Pharmacy, USC Alfred E. Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, 90089, United States.
USC Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, 90089, United States.
Biol Methods Protoc. 2024 Sep 5;9(1):bpae064. doi: 10.1093/biomethods/bpae064. eCollection 2024.
Characterization of T-cell receptors (TCRs) repertoire was revolutionized by next-generation sequencing technologies; however, standardization using biological controls to facilitate precision of current alignment and assembly tools remains a challenge. Additionally, availability of TCR libraries for off-the-shelf cloning and engineering TCR-specific T cells is a valuable resource for TCR-based immunotherapies. We established nine human TCR α and β clones that were evaluated using the 5'-rapid amplification of cDNA ends-like RNA-based TCR sequencing on the Illumina platform. TCR sequences were extracted and aligned using MiXCR, TRUST4, and CATT to validate their sensitivity and specificity and to validate library preparation methods. The correlation between actual and expected TCR ratios within libraries confirmed accuracy of the approach. Our findings established the development of biological standards and library of TCR clones to be leveraged in TCR sequencing and engineering. The remaining human TCR clones' libraries for a more diverse biological control will be generated.
下一代测序技术彻底改变了T细胞受体（TCR）库的表征；然而，使用生物对照进行标准化以提高当前比对和组装工具的精度仍然是一项挑战。此外，用于现成克隆和工程化TCR特异性T细胞的TCR文库的可用性是基于TCR的免疫疗法的宝贵资源。我们建立了9个人类TCR α和β克隆，并在Illumina平台上使用基于5'-cDNA末端快速扩增样RNA的TCR测序进行了评估。使用MiXCR、TRUST4和CATT提取并比对TCR序列，以验证它们的敏感性和特异性，并验证文库制备方法。文库中实际和预期TCR比率之间的相关性证实了该方法的准确性。我们的研究结果确立了生物标准和TCR克隆文库的开发，可用于TCR测序和工程。将生成其余人类TCR克隆文库，用于更多样化的生物对照。
