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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Decombinator: a tool for fast, efficient gene assignment in T-cell receptor sequences using a finite state machine
TL;DR: A simple five-item identifier is defined that uniquely and unambiguously defines each TcR sequence and a novel application of finite-state automaton is described to map Illumina short-read sequence data for individual TcRs to their respective identifier.
Journal ArticleDOI
IMonitor: A Robust Pipeline for TCR and BCR Repertoire Analysis
Wei Zhang,Du Yuanping,Zheng Su,Changxi Wang,Xiaojing Zeng,Ruifang Zhang,Hong Xueyu,Chao Nie,Jinghua Wu,Hongzhi Cao,Xun Xu,Xiao Liu +11 more
TL;DR: A methodology is developed to correct the PCR and sequencing errors and to minimize the PCR bias among various rearranged sequences with different V and J gene families, and this package would be of widespread usage for immune repertoire analysis.
Patent
Monitoring health and disease status using clonotype profiles
Malek Faham,Thomas D. Willis +1 more
TL;DR: In this article, a method for using DNA sequencing to identify personalized, or patient-specific biomarkers in patients with lymphoid neoplasms, autoimmune disease and other conditions is presented.
Journal ArticleDOI
T cells, aging and senescence.
Luca Pangrazzi,Birgit Weinberger +1 more
TL;DR: It has been postulated that highly differentiated senescent-like T cells are unable to eliminate other senescent cell types, and a reliable definition of senescent T cells is essential for an extension of this concept to T cells.
Journal ArticleDOI
High-throughput sequencing of the T-cell receptor repertoire: pitfalls and opportunities.
TL;DR: This work outlines the major steps in processing of repertoire data, considering low‐level processing of raw sequence files and high‐level algorithms, which seek to extract biological or pathological information from T‐cell receptor data.
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