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ABSTRACT The T cell compartment must contain diversity in both TCR repertoire and cell state to provide effective immunity against pathogens 1,2 . However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state at the single cell level because most analysis of the TCR repertoire has, to date, aggregated information from populations of cells. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current protocols to directly sequence the TCR require the use of long sequencing reads, increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present a tool that can efficiently extract TCR sequence information from standard, short-read scRNA-seq libraries of T cells: TCR Reconstruction Algorithm for Paired-End Single cell (TRAPeS). We apply it to investigate heterogeneity in the CD8 + T cell response in humans and mice, and show that it is accurate and more sensitive than previous approaches 3,4 . We applied TRAPeS to single cell RNA-seq of CD8 + T cells specific for a single epitope from Yellow Fever Virus 5 . We show that the recently-described "naive-like" memory population of YFV-specific CD8 + T cells have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype CD8 + T cells specific for YFV. This suggests that TCR usage contributes to heterogeneity in the differentiation state of the CD8 + T cell response to YFV. TRAPeS is publicly available, and can be readily used to investigate the relationship between the TCR repertoire and cellular phenotype.

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