Exhaustive T-cell repertoire sequencing of human peripheral blood samples reveals signatures of antigen selection and a directly measured repertoire size of at least 1 million clonotypes
René L. Warren,J. Douglas Freeman,Thomas Zeng,Gina Choe,Sarah A. Munro,Richard D. Moore,John R. Webb,Robert A. Holt +7 more
TLDR
It is shown that the sensitivity of sequence-based repertoire profiling is limited by both sequencing depth and sequencing accuracy, and a new, directly measured, lower limit on individual T-cell repertoire size is established.Abstract:
Massively parallel sequencing is a useful approach for characterizing T-cell receptor diversity. However, immune receptors are extraordinarily difficult sequencing targets because any given receptor variant may be present in very low abundance and may differ legitimately by only a single nucleotide. We show that the sensitivity of sequence-based repertoire profiling is limited by both sequencing depth and sequencing accuracy. At two timepoints, 1 wk apart, we isolated bulk PBMC plus naive (CD45RA+/CD45RO-) and memory (CD45RA-/CD45RO+) T-cell subsets from a healthy donor. From T-cell receptor beta chain (TCRB) mRNA we constructed and sequenced multiple libraries to obtain a total of 1.7 billion paired sequence reads. The sequencing error rate was determined empirically and used to inform a high stringency data filtering procedure. The error filtered data yielded 1,061,522 distinct TCRB nucleotide sequences from this subject which establishes a new, directly measured, lower limit on individual T-cell repertoire size and provides a useful reference set of sequences for repertoire analysis. TCRB nucleotide sequences obtained from two additional donors were compared to those from the first donor and revealed limited sharing (up to 1.1%) of nucleotide sequences among donors, but substantially higher sharing (up to 14.2%) of inferred amino acid sequences. For each donor, shared amino acid sequences were encoded by a much larger diversity of nucleotide sequences than were unshared amino acid sequences. We also observed a highly statistically significant association between numbers of shared sequences and shared HLA class I alleles.read more
Citations
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Imgt/highv-quest: the imgt® web portal for immunoglobulin (ig) or antibody and t cell receptor (tr) analysis from ngs high throughput and deep sequencing
TL;DR: The challenge was to provide identical high quality analysis for the huge number of sequences obtained by Next Generation Sequencing (NGS) high throughput and deep sequencing.
Journal ArticleDOI
Systems immunology: just getting started.
TL;DR: Strategies for measuring a broad swath of immunological functions as a means of discovering previously unknown relationships and phenomena and as a powerful way of understanding the immune system as a whole are reviewed.
Journal ArticleDOI
MiTCR: software for T-cell receptor sequencing data analysis
Dmitriy A. Bolotin,Mikhail Shugay,Ilgar Z. Mamedov,Ekaterina V. Putintseva,Maria A. Turchaninova,Ivan V. Zvyagin,Ivan V. Zvyagin,Olga V. Britanova,Dmitriy M. Chudakov,Dmitriy M. Chudakov +9 more
TL;DR: The accuracy and specificity of MiTCR for the analysis of both cDNA-based and genomic DNA–based high-throughput sequencing datasets is demonstrated and the efficiency of human TCR-α and T CR-β CDR3 extraction, clonotype generation and error correction for the model data is determined.
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Dietary gluten triggers concomitant activation of CD4+ and CD8+ αβ T cells and γδ T cells in celiac disease
Arnold Han,Evan W. Newell,Jacob Glanville,Nielsen Fernandez-Becker,Chaitan Khosla,Yueh-hsiu Chien,Mark M. Davis +6 more
TL;DR: A previously unappreciated role of antigen is revealed in the induction of CD8+ αβ and γδ T cells in celiac disease and a coordinated response by all three of the major types of T cells is demonstrated.
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The Past, Present, and Future of Immune Repertoire Biology – The Rise of Next-Generation Repertoire Analysis
Adrien Six,Maria Encarnita Mariotti-Ferrandiz,Wahiba Chaara,Susana Magadán,Hang-Phuong Pham,Marie-Paule Lefranc,Thierry Mora,Véronique Thomas-Vaslin,Aleksandra M. Walczak,Pierre Boudinot +9 more
TL;DR: A broad range of available methodological tools developed in the past decades, each of which answering different questions and showing complementarity for progressive identification of the level of repertoire alterations, can be found in this article.
References
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