The gremlin in the works: why T cell receptor researchers need to pay more attention to germline reference sequences

<h2>Abstract</h2> The rise of T cell receptor (TCR) sequencing technologies is driving both new understandings of the immune system and the development of novel clinical platforms. Such analyses rely on comparing recombined TCR sequences to unrearranged germline reference sequences during V(D)J annotation. In this study, we observed that despite the importance of this step in TCR analysis, most published studies do not properly report the reference used. We use public datasets to illustrate why references should be explicitly specified: using IMGT/GENE-DB as an example, we document how the reference set changes over time. Furthermore we illustrate how prescriptivist interpretations of reference metadata may be obscuring rather than illuminating TCR biology, and demonstrate the need to perform full V gene sequencing in order to unambiguously determine the final translated TCR polypeptide sequence. In summary, we argue that in order to ensure the accuracy and reproducibility of TCR sequencing – an ever more pressing task as more TCR-based diagnostics and therapeutics are developed – we should all take more care with the development, use, and reporting of the TCR germline references used in our science.