Quantifying The Impact of Bulk TCR-Seq Methodological Choices on The Profiled T Cell Repertoire
Bulk T cell repertoire profiling using sequencing (TCR-Seq) is a powerful method to investigate T cell responses to natural infections, vaccines, cancers, and autoimmune diseases. This assay can be conducted using various techniques, such as multiplex PCR or 5-RACE. However, each methods introduces systematic biases that can result in different pictures of the underlying T cell repertoire. Furthermore, the impact of technical variables on the accuracy of these methods remains understudied. Thus, in this study, we systematically characterized different multiplex PCR-based protocols, focusing on the quality and quantity of the utilized RNA/DNA, extraction methods, amplification programs, variations between production batches, and technical handling of samples. Our findings highlight the important role of RNA/DNA quality in shaping the profiling results of T cell repertoires. Whereas low RNA/DNA quantities can be partially compensated for by increasing the number of PCR cycles, this is partially not possible with lower quality. In conclusion, our results highlight the influence of different technical choices on the biological conclusions drawn from TCR-Seq data and provide practical guidelines to finetune these variables to ensure consistent and reliable results under diverse experimental constraints.