A SMARTer approach to profiling the human T-cell receptor repertoire

Abstract

Abstract Profiling T-cell receptor (TCR) repertoires involves characterizing the diversity of TCR nucleotide sequences in a sample, and is an increasingly popular approach for analyzing the composition of the adaptive immune system. While low-throughput approaches have yielded important insights concerning TCR repertoire dynamics, development of next-generation sequencing (NGS) technologies has dramatically expanded research prospects. Using SMART® technology, we have developed an NGS library preparation kit for TCR profiling that employs a 5′ RACE-like approach to capture full-length variable regions of TCR-α and/or TCR-β subunits. With this approach, 10 ng–3 μg of input RNA or 50–10,000 purified T cells undergo reverse transcription using dT priming. Following first-strand cDNA synthesis, semi-nested PCR is used to amplify TCR-specific sequences and add Illumina® sequencing adapters. Starting with peripheral blood RNA, libraries containing TCR-α and TCR-β sequences were generated and sequenced. For each replicate >70% of reads were on-target, and the most highly represented clonotypes remained consistent across a range of input amounts. A separate sensitivity assay demonstrated that RNA corresponding to a single clonotype could be detected above background levels when spiked into input RNA at a relative concentration of 0.1 %. Our approach enables the user to obtain sequencing-ready TCR libraries from RNA in ~2.5 hours of hands-on time. Whereas approaches that utilize genomic DNA as starting material require multiplexed PCR strategies, RNA based amplification of TCR sequences use single primer pairs for each subunit. In this way, our approach minimizes the likelihood of sample misrepresentation due to amplification biases.