ab14cf6ccc6dd95538fb61fa9e501ee2 328235.pdf 590f493e2c2acc72910df0f0dbf411269d7463df 328235.pdf c14751f2440403e00d0826cf2cbf4e2c19cc70cdcf88fada13b632d89b86915c 328235.pdf Title: A High-Throughput Sequencing Strategy for Clinical Repertoire Profiling of T Cell Receptor Beta Chain: Development and Reference Values Across Healthy Adults, Paediatrics, and Cord Blood Units Subject: T cell receptor (TCR) profiling using next-generation sequencing (NGS) enables high-throughput, in-depth analysis of repertoire diversity, offering numerous clinical applications. We developed a DNA-based strategy to analyse the TCR-chain using NGS and established reference values for T cell repertoire characteristics in 74 healthy donors, including 44 adults, 20 paediatrics, and 10 cord blood units (CBUs). Additionally, four paediatric patients with combined immunodeficiency (CID) or severe CID (SCID) due to deleterious mutations in recombination activating genes (RAG) were analysed. The developed strategy demonstrated high specificity, reproducibility, and sensitivity, and all functional variable and joining genes were detected with minimal PCR bias. All donors had a Gaussian-like distribution of complementary-determining region 3 length, with lower presence of non-templated nucleotides and higher proportion of non-functional clonotypes in CBUs. Both CBUs and paediatrics showed greater convergence and TCR diversity was significantly lower in adults and donors with cytomegalovirus-positive serostatus. Finally, an analysis of paediatric patients with RAG-SCID/CID showed significantly shorter CDR3 region length and lower repertoire diversity compared to healthy paediatrics. In summary, we developed a reliable and feasible TCR sequencing strategy for application in the clinical setting, and established reference values that could assist in the diagnosis and monitoring of pathological conditions affecting the T cell repertoire. Keywords: T cell receptor; next-generation sequencing analysis; molecular methodologies; molecular diagnostic Author: Emma Enrich, Mireia Antón-Iborra, Carlos Hobeich, Rut Mora-Buch, Ana Gabriela Lara-de-León, Alba Parra-Martínez, Belén Sánchez, Francisco Vidal, Pere Soler-Palacin and Francesc Rudilla Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Wed Oct 1 11:34:21 2025 CEST ModDate: Wed Oct 1 11:38:18 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3285055 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IQEKBJ+URWPalladioL-Ital Type 1 Custom yes yes yes 125 0 AIRHKL+VnURWPalladioL Type 1 Custom yes yes yes 126 0 TWLYIF+URWPalladioL-Bold Type 1 Custom yes yes yes 127 0 IYIESL+URWPalladioL-Roma Type 1 Custom yes yes yes 128 0 KYGVAL+EURM10 Type 1 Builtin yes yes yes 129 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes yes 171 0 YZIJXT+CMSY10 Type 1 Builtin yes yes yes 172 0 CIDFont+F2 CID TrueType Identity-H yes no yes 209 0 CIDFont+F4 CID TrueType Identity-H yes no yes 210 0 CIDFont+F2 CID TrueType Identity-H yes no yes 224 0 CIDFont+F2 CID TrueType Identity-H yes no yes 264 0 CIDFont+F4 CID TrueType Identity-H yes no yes 265 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 329 0 CIDFont+F3 CID TrueType Identity-H yes no yes 315 0 CIDFont+F2 CID TrueType Identity-H yes no yes 316 0 CIDFont+F4 CID TrueType Identity-H yes no yes 317 0 CIDFont+F3 CID TrueType Identity-H yes no yes 322 0 CIDFont+F2 CID TrueType Identity-H yes no yes 323 0 CIDFont+F4 CID TrueType Identity-H yes no yes 324 0 CIDFont+F2 CID TrueType Identity-H yes no yes 359 0 CIDFont+F4 CID TrueType Identity-H yes no yes 360 0 CIDFont+F3 CID TrueType Identity-H yes no yes 396 0 CIDFont+F2 CID TrueType Identity-H yes no yes 397 0 CIDFont+F4 CID TrueType Identity-H yes no yes 398 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-06-19 04:34:41 CEST RepresentationInformation: 328235.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-06-18 14:40:13 CEST Size: 3285055 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 638 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results Children: Item: Title: TCR NGS Strategy Item: Title: Primer Specificity and Efficiency Item: Title: Reproducibility and Sensitivity Item: Title: Establishment of Working Samples Item: Title: Reference Values for TCR Repertoire Profiling Item: Title: TCR Repertoire Profiling in RAG-SCID/CID Patients Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Samples and Ethics Committee Item: Title: CD3+ T Cell Quantification Using Flow Cytometry Item: Title: Primer Design and Optimisation Item: Title: TCR Repertoire Profiling in Healthy Donors and Patients with RAG-SCID/CID Item: Title: Sequencing Analysis Item: Title: Statistical Analysis Item: Title: References Info: Title: A High-Throughput Sequencing Strategy for Clinical Repertoire Profiling of T Cell Receptor Beta Chain: Development and Reference Values Across Healthy Adults, Paediatrics, and Cord Blood Units Author: Emma Enrich, Mireia Antón-Iborra, Carlos Hobeich, Rut Mora-Buch, Ana Gabriela Lara-de-León, Alba Parra-Martínez, Belén Sánchez, Francisco Vidal, Pere Soler-Palacin and Francesc Rudilla Subject: T cell receptor (TCR) profiling using next-generation sequencing (NGS) enables high-throughput, in-depth analysis of repertoire diversity, offering numerous clinical applications. We developed a DNA-based strategy to analyse the TCR-chain using NGS and established reference values for T cell repertoire characteristics in 74 healthy donors, including 44 adults, 20 paediatrics, and 10 cord blood units (CBUs). Additionally, four paediatric patients with combined immunodeficiency (CID) or severe CID (SCID) due to deleterious mutations in recombination activating genes (RAG) were analysed. The developed strategy demonstrated high specificity, reproducibility, and sensitivity, and all functional variable and joining genes were detected with minimal PCR bias. All donors had a Gaussian-like distribution of complementary-determining region 3 length, with lower presence of non-templated nucleotides and higher proportion of non-functional clonotypes in CBUs. Both CBUs and paediatrics showed greater convergence and TCR diversity was significantly lower in adults and donors with cytomegalovirus-positive serostatus. Finally, an analysis of paediatric patients with RAG-SCID/CID showed significantly shorter CDR3 region length and lower repertoire diversity compared to healthy paediatrics. In summary, we developed a reliable and feasible TCR sequencing strategy for application in the clinical setting, and established reference values that could assist in the diagnosis and monitoring of pathological conditions affecting the T cell repertoire. 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