Uddin, I;
Joshi, K;
Oakes, T;
Heather, JM;
Swanton, C;
TRACERx consortium, .;
Chain, B;
(2019)
An Economical, Quantitative, and Robust Protocol for High-Throughput T Cell Receptor Sequencing from Tumor or Blood.
Cancer Immunosurveillance: Methods and Protocols
pp. 15-42.
10.1007/978-1-4939-8885-3_2.
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Abstract
The T cell receptor repertoire provides a window to the cellular adaptive immune response within a tumor, and has the potential to identify specific and personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing, and analyzing T cell receptors which is economical, robust, sensitive, and versatile. The key experimental step is the ligation of a single-stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. This method has been applied to the analysis of unfractionated human tumor lysates, subpopulations of tumor-infiltrating lymphocytes, and peripheral blood samples from patients with a variety of solid tumors.
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