Benchmarking of T cell Receptor repertoire profiling methods reveals large systematic biases

Monitoring the T cell receptor (TCR) repertoire is crucial for understanding immune responses, but the accuracy of current TCR sequencing (TCRseq) methods is uncertain. This study compared nine TCRseq methods, including six RACE-PCR and three multiplex-PCR approaches, on the same T cell sample. Significant differences were found in accuracy and reproducibility for TCR α (TRA) and β (TRB) chains.

Most methods had difficulty capturing TRA diversity, especially with low RNA input. 5′ RACE-PCR methods were consistent with each other but differed from RNA-based multiplex-PCR results. A genomic DNA-based method and two non-UMI RNA-based methods were more sensitive in detecting rare clonotypes, although UMI methods were more accurate in clonotype quantification.