Abstract
The T cell receptor (TCR) repertoire can provide a personalized biomarker for infectious and non-infectious diseases. We describe a protocol for amplifying, sequencing, and analyzing TCRs which is robust, sensitive, and versatile. The key experimental step is ligation of a single-stranded oligonucleotide to the 3′ end of the TCR cDNA. This allows amplification of all possible rearrangements using a single set of primers per locus. It also introduces a unique molecular identifier to label each starting cDNA molecule. This molecular identifier is used to correct for sequence errors and for effects of differential PCR amplification efficiency, thus producing more accurate measures of the true TCR frequency within the sample. This integrated experimental and computational pipeline is applied to the analysis of human memory and naive subpopulations, and results in consistent measures of diversity and inequality. After error correction, the distribution of TCR sequence abundance in all subpopulations followed a power law over a wide range of values. The power law exponent differed between naïve and memory populations, but was consistent between individuals. The integrated experimental and analysis pipeline we describe is appropriate to studies of T cell responses in a broad range of physiological and pathological contexts.
Highlights
The adaptive immune system of jawed vertebrates uses imprecise somatic DNA recombination to generate a rich and diverse array of antigen specific receptors on B cells (BCR) and T cells (TCR)
The mechanisms for the generation of diversity have been studied in great detail, the very diversity of the sequences coding for the receptors prevented a global analysis of the repertoire of B or T cell antigen receptors using conventional DNA sequencing techniques
We present an integrated experimental and computational pipeline for T cell receptor (TCR) repertoire analysis and use it to provide a quantitative description of the repertoire in different sorted populations of human memory and naïve T cells
Summary
The adaptive immune system of jawed vertebrates uses imprecise somatic DNA recombination to generate a rich and diverse array of antigen specific receptors on B cells (BCR) and T cells (TCR). The mechanisms for the generation of diversity have been studied in great detail, the very diversity of the sequences coding for the receptors prevented a global analysis of the repertoire of B or T cell antigen receptors using conventional DNA sequencing techniques. Repertoire analysis provides many experimental and computational challenges, for which various solutions have been proposed [4, 6,7,8]. Logistic considerations such as cost, ease of use, robustness, and versatility, as well as more scientific issues such as accuracy and coverage, may contribute to which solution is optimal for different laboratories
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
