replicateFest: an R package and shiny app for analysis of T cell receptor repertoire data from the functional...

The Functional Expansion of Specific T cell (FEST)-based assays combine short-term peptide stimulation with TCR sequencing to identify clonotypes that expand in response to specific antigens. These approaches have proven invaluable for detecting antigen-specific T cell responses, guiding vaccine development, and assessing checkpoint blockade efficacy. However, variability introduced by biological and technical replicates poses challenges for reproducibility and interpretation, and existing computational tools do not address replicate-level analysis in these assays.

We developed replicateFest, a computational framework implemented as an R package and Shiny web application, to analyze FEST-based TCR-seq data with and without replicates. replicateFest applies Fisher's exact test for non-replicate datasets and negative binomial modeling for replicate experiments, returning adjusted p-values and odds ratios to identify clonotypes significantly expanded in antigen-stimulated conditions. The framework distinguishes FEST-expanded clonotypes (relative to a no-antigen control) and FEST-positive clonotypes (expanded compared to all other conditions). Validation using in silico datasets confirmed accurate detection of antigen-specific clonotypes. Application to published HIV-1 epitope stimulation data reproduced original findings and demonstrated replicateFest's utility for reproducibility assessment and quality control.