{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:33:13Z","timestamp":1785407593471,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,23]]},"group-title":"Immunology","reference-count":13,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["P30CA006973"],"award-info":[{"award-number":["P30CA006973"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R37CA251447"],"award-info":[{"award-number":["R37CA251447"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R50CA243627"],"award-info":[{"award-number":["R50CA243627"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P50CA098252"],"award-info":[{"award-number":["P50CA098252"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100016947","name":"Swim Across America","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100016947","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100004436","name":"Commonwealth Foundation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100004436","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Motivation<\/jats:title>\n                  <jats:p>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 neoantigen-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.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>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\u2019s 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 synthetic datasets confirmed accurate detection of antigen-specific clonotypes. Application to published HIV-1 epitope stimulation data reproduced original findings and demonstrated replicateFest\u2019s utility for reproducibility assessment and quality control.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Availability and Implementation<\/jats:title>\n                  <jats:p>\n                    replicateFest is freely available under the Apache-2.0 license as an R package at\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/github.com\/OncologyQS\/replicateFest\">https:\/\/github.com\/OncologyQS\/replicateFest<\/jats:ext-link>\n                    and as an interactive Shiny application at\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/www.stat-apps.onc.jhmi.edu\/FEST\/\">http:\/\/www.stat-apps.onc.jhmi.edu\/FEST\/<\/jats:ext-link>\n                    .\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.18.733036","type":"posted-content","created":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T22:50:24Z","timestamp":1782255024000},"source":"Crossref","is-referenced-by-count":0,"title":["replicateFest: An R Package and Shiny App for Analysis of T Cell Receptor Repertoire Data from the Functional Expansion"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2813-3094","authenticated-orcid":false,"given":"Ludmila","family":"Danilova","sequence":"first","affiliation":[{"name":"Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA"},{"name":"Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins School of Medicine, Baltimore, MD, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2595-1091","authenticated-orcid":false,"given":"Alexander V.","family":"Favorov","sequence":"additional","affiliation":[{"name":"Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kellie N.","family":"Smith","sequence":"additional","affiliation":[{"name":"Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA"},{"name":"Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins School of Medicine, Baltimore, MD, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4098-3289","authenticated-orcid":false,"given":"Leslie","family":"Cope","sequence":"additional","affiliation":[{"name":"Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026071510101909000_2026.06.18.733036v3.1","doi-asserted-by":"publisher","DOI":"10.1158\/2326-6066.CIR-18-0129"},{"key":"2026071510101909000_2026.06.18.733036v3.2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3364"},{"key":"2026071510101909000_2026.06.18.733036v3.3","unstructured":"Home - immunomind. [Internet]. [cited 2025 Nov 21]. Available from: https:\/\/immunomind.github.io\/docs\/"},{"key":"2026071510101909000_2026.06.18.733036v3.4","unstructured":"Mangul-Lab-USC\/reTCR [Internet]. Mangul Lab at USC; 2024 [cited 2025 Nov 21]. Available from: https:\/\/github.com\/Mangul-Lab-USC\/reTCR"},{"issue":"4","key":"2026071510101909000_2026.06.18.733036v3.5","doi-asserted-by":"crossref","first-page":"qzae013","DOI":"10.1093\/gpbjnl\/qzae013","article-title":"TCRosetta: An Integrated Analysis and Annotation Platform for T-cell Receptor Sequences","volume":"22","year":"2024","journal-title":"Genomics Proteomics Bioinformatics"},{"key":"2026071510101909000_2026.06.18.733036v3.6","doi-asserted-by":"crossref","unstructured":"Perik-Zavodskii R , Perik-Zavodskaia O , Volynets M , Alrhmoun S , Sennikov S. TCRscape: a single-cell multi-omic TCR profiling toolkit. Front Bioinform [Internet]. 2025 Sep 5 [cited 2025 Nov 21];5. Available from: https:\/\/www.frontiersin.org\/journals\/bioinformatics\/articles\/10.3389\/fbinf.2025.1641491\/full","DOI":"10.3389\/fbinf.2025.1641491"},{"key":"2026071510101909000_2026.06.18.733036v3.7","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.3000314"},{"key":"2026071510101909000_2026.06.18.733036v3.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1808594115"},{"key":"2026071510101909000_2026.06.18.733036v3.9","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-2712-9_15"},{"key":"2026071510101909000_2026.06.18.733036v3.10","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1007\/978-1-0716-2712-9_16","article-title":"Flexible Distance-Based TCR Analysis in Python with tcrdist3","volume":"2574","year":"2022","journal-title":"Methods Mol Biol"},{"key":"2026071510101909000_2026.06.18.733036v3.11","doi-asserted-by":"crossref","unstructured":"Perik-Zavodskii R , Perik-Zavodskaia O , Volynets M , Alrhmoun S , Sennikov S. 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