T-cell receptor (TCR) profiling by next-generation sequencing
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1
Mcgill university, Departments of Pediatrics and Human Genetics, Canada
Background
T-lymphocytes have the capacity to recognize antigens from millions of pathogens with a remarkable degree of specificity, due to the molecular diversity of the T cell receptor (TCR) repertoire. Because of this diversity, the TCR, arguably the most important molecule in both host defense and autoimmunity has been impossible to study in-depth with conventional sequencing. Next-generation sequencing, by defining individual molecules, now allows the delineation of this repertoire in unprecedented depth.
Objective
To evaluate the accuracy of our innovative TCR profiling protocol.
Experimental Approach
RNA from T cells clones with one defined TCR rearrangement was mixed at different concentrations with samples of normal human CD4+ T cells. A 5’-RACE protocol that we have developed was used for the unbiased amplification of all TCRs, despite the large diversity of their 5' ends. Amplicons were multiplexed and sequenced at a depth of 1,000,000x on the Illumina MiSeq. V, J and D segment choice were assigned by a paired-ends modification of the IMGT algorithm. Copy count of TCR clones was used to calculate the frequency of each clone in every mix.
Results
In our preliminary studies the assay was able to accurately detect the specific clones as well as their concentrations in each mix. It was found to be sensitive, reliable and repeatable.
Conclusions
Accurate profiling of the TCR repertoire using 5’ race and next generation sequencing is a promising unbiased and precise method.
Keywords:
TCR Profiling,
5' RACE,
next generation sequencing,
TCR repertoire,
T-cell receptor
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Immune receptors and signaling
Citation:
Oron
T,
Marchand
L,
Li
Q and
Polychronakos
C
(2013). T-cell receptor (TCR) profiling by next-generation sequencing.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.01171
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Received:
22 Jul 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Dr. Tal Oron, Mcgill university, Departments of Pediatrics and Human Genetics, Montreal, Quebec, H3Z 2Z3, Canada, orotal@gmail.com