scispace - formally typeset
Open AccessJournal ArticleDOI

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

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

Content maybe subject to copyright    Report

Citations
More filters
Journal ArticleDOI

Derivation of HLA types from shotgun sequence datasets

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.
Journal ArticleDOI

Sequence analysis of T-cell repertoires in health and disease

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.
References
More filters
Journal ArticleDOI

Clustal W and Clustal X version 2.0

TL;DR: The Clustal W and ClUSTal X multiple sequence alignment programs have been completely rewritten in C++ to facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems.
Journal ArticleDOI

Base-calling of automated sequencer traces using Phred. I. accuracy assessment

TL;DR: In this article, a base-calling program for automated sequencer traces, phred, with improved accuracy was proposed. But it was not shown to achieve a lower error rate than the ABI software, averaging 40%-50% fewer errors in the data sets examined independent of position in read, machine running conditions, or sequencing chemistry.
Journal ArticleDOI

Base-Calling of Automated Sequencer Traces Using Phred. II. Error Probabilities

TL;DR: The ability to estimate a probability of error for each base-call, as a function of certain parameters computed from the trace data, is developed and implemented in the base-calling program.
Journal ArticleDOI

T-cell antigen receptor genes and T-cell recognition.

TL;DR: This view of T-cell recognition has implications for how the receptors might be selected in the thymus and how they (and immunoglobulins) may have arisen during evolution.
Related Papers (5)