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
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Journal ArticleDOI
Derivation of HLA types from shotgun sequence datasets
René L. Warren,Gina Choe,Douglas J Freeman,Mauro Castellarin,Sarah Munro,Richard A. Moore,Robert A. Holt,Robert A. Holt +7 more
TL;DR: HLAminer, a computational method for identifying HLA alleles directly from shotgun sequence datasets that circumvents the additional time and cost of generating HLA-specific data and capitalizes on the increasing accessibility and affordability of massively parallel sequencing.
Posted ContentDOI
A cell atlas of human thymic development defines T cell repertoire formation
Jong-Eun Park,Rachel A. Botting,Cecilia Domínguez Conde,Dorin-Mirel Popescu,Marieke Lavaert,Daniel J Kunz,Daniel J Kunz,Daniel J Kunz,Emily Stephenson,Roberta Ragazzini,Elizabeth Tuck,Anna Wilbrey-Clark,John R. Ferdinand,Simone Webb,Daniel Maunder,Niels Vandamme,Krishnaa T. Mahbubani,Krzysztof Polanski,Lira Mamanova,Andrew Fuller,Andrew Filby,Gary Reynolds,David Dixon,Kourosh Saeb-Parsy,Steven Lisgo,Deborah J. Henderson,Roser Vento-Tormo,Kerstin B. Meyer,Yvan Saeys,Paola Bonfanti,Paola Bonfanti,Sam Behjati,Menna R. Clatworthy,Menna R. Clatworthy,Tom Taghon,Muzlifah Haniffa,Muzlifah Haniffa,Sarah A. Teichmann,Sarah A. Teichmann +38 more
TL;DR: A bias in TCR recombination and selection is revealed, which is attributed to genomic position and suggests later commitment of the CD8+ T-cell lineage, and a comprehensive atlas of the human thymus across the lifespan is provided.
Journal ArticleDOI
Sequence analysis of T-cell repertoires in health and disease
Daniel J Woodsworth,Daniel J Woodsworth,Mauro Castellarin,Mauro Castellarin,Robert A. Holt,Robert A. Holt,Robert A. Holt +6 more
TL;DR: T-cell antigen receptor (TCR) variability enables the cellular immune system to discriminate between self and non-self as mentioned in this paper, and T-cell repertoire sequencing is still in its infancy, however, it is expected that maturation of the field will involve the introduction of improved, standardized tools for data handling, deposition and statistical analysis, as well as the emergence of new and equivalently large-scale technologies for Tcell functional analysis and antigen discovery.
Journal ArticleDOI
Bioinformatic and Statistical Analysis of Adaptive Immune Repertoires.
TL;DR: The current literature on bioinformatic and statistical analysis of immune repertoire HTS data is reviewed and important directions for future research are highlighted, which could propel immune repertoires HTS to becoming a standard method for measuring adaptive immune responses.
Journal ArticleDOI
Estimating the Diversity, Completeness, and Cross-Reactivity of the T Cell Repertoire
TL;DR: It is shown that the existing hypotheses fail to explain why the immune system has the potential to generate far more diversity than is used in an individual, and an alternative hypothesis of “evolutionary sloppiness” is proposed.
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