![]() Low abundance and may differ legitimately from other receptor variants by only a single nucleotide. Immune receptors are extraordinarily difficult sequencing targets because any given receptor variant may be present in very Recognition and mitigation of sequence error is essential for accurate repertoire enumeration All data from the present study have been made available as a community resource to facilitateįuture comparative studies of immune repertoires. Still, immune repertoire analysis by massively parallel sequencing offers tremendous utility, where distinct clonotypes canīe readily identified and tracked, diversity can be profiled, and differences among individuals or subsets of sorted cellsĬan be readily distinguished. Sequencing errors are incurred at a constant rate, it is not possible to distinguish very rare sequences from sequencing errors. ![]() Intractable because a repertoire can only be subsampled, and by the nature of “next-generation” sequencing technologies where However, determining the true size of an immune repertoire by exhaustive sequencing is We find that by exhaustive sequencingĪnd careful mitigation of sequencing error, it is possible to saturate the diversity within a single sequencing library and Here, we analyze TCR beta-chain sequences from peripheral blood from a single healthy individual,Īnd we compare this immune repertoire to survey sequence from two other healthy individuals. ![]() Sequence diversity in both T-cell and B-cell immune repertoires has been surveyed previously ( Boyd et al. We use the sequence of the CDR3 plus the identity of the flanking V and J gene segments to uniquely classify TCRB variants. Portion of the TCR that directly contacts pMHC ( Davis and Bjorkman 1988). Is within the CDR3 (Complementarity Determining Region 3), which encompasses the VDJ recombination junctions and encodes the 2004 Harty and Badovinac 2008).īy allelic exclusion, a T cell typically expresses only a single TCRB variant ( Khor and Sleckman 2002), making beta-chain sequence variation a useful measure of T-cell repertoire diversity. Is restricted by deletion of over- and under-reactive cells during thymic maturation and is molded continuously by the clonalĮxpansion of antigen responsive cells in the periphery ( Nikolich-Zugich et al. Like antibody diversity, the potential for TCR diversity is nearly infinite, but actual diversity in a biological repertoire The process is directly analogous to the generation of antibody diversity by somatic VDJ recombination of the B-cell receptor Junctions ( Davis and Bjorkman 1988 Bassing et al. The Variable (V), Diversity (D), and Joining (J) gene segments, plus the addition/subtraction of nontemplated bases at recombination Of recognizing diverse peptide–MHC (pMHC) complexes, the locus encoding the receptor undergoes somatic recombination among To generate a repertoire of structurally variant TCRs capable Of that cell by the major histocompatibility complex (MHC). Surface of a T lymphocyte will bind to a protein degradation product from the heterologous cell that is presented at the surface Recognition is mediated by the interaction of cell surface molecules, whereby a heterodimeric T-cell receptor (TCR) on the T lymphocytes are key mediators of adaptive immunity that recognize heterologous cells expressing foreign or mutated proteins. We also observed a highly statistically significant association between numbers of shared sequences To 1.1%) of nucleotide sequences among donors, but substantially higher sharing (up to 14.2%) of inferred amino acid sequences.įor each donor, shared amino acid sequences were encoded by a much larger diversity of nucleotide sequences than were unsharedĪmino acid sequences. Sequences obtained from two additional donors were compared to those from the first donor and revealed limited sharing (up On individual T-cell repertoire size and provides a useful reference set of sequences for repertoire analysis. Yielded 1,061,522 distinct TCRB nucleotide sequences from this subject which establishes a new, directly measured, lower limit The sequencingĮrror rate was determined empirically and used to inform a high stringency data filtering procedure. MRNA we constructed and sequenced multiple libraries to obtain a total of 1.7 billion paired sequence reads. (CD45RA+/CD45RO−) and memory (CD45RA−/CD45RO+) T-cell subsets from a healthy donor. At two timepoints, 1 wk apart, we isolated bulk PBMC plus naïve Limited by both sequencing depth and sequencing accuracy. We show that the sensitivity of sequence-based repertoire profiling is May differ legitimately by only a single nucleotide. However, immune receptorsĪre extraordinarily difficult sequencing targets because any given receptor variant may be present in very low abundance and Massively parallel sequencing is a useful approach for characterizing T-cell receptor diversity. ![]()
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