High-throughput pairing of T cell receptor alpha and beta sequences (TECH2P.930)

Conventional high-throughput immunosequencing methods profile TCR alpha (TRA) and/or TCR beta (TRB) repertoires, but do not enable pairing of the cognate subunits comprising functional TCRs. We developed a method called pairSEQ that can pair hundreds of thousands of TRA and TRB sequences in a single experiment. Our approach works by allocating random samples of T cells to a micro-titer plate, sequencing amplified cDNA from TRA and TRB genes, and DNA bar-coding the wells. Most TCR sequences uniquely identify clonal cell populations, so pairs can be assigned based on TRA and TRB sequences that occupy the same wells. To validate our method we identified cognate pairs from a sample of cells mixed from two people. The cross-sample pairs directly measure false pairing rates, which closely matched predictions from our statistical model. To show our method can be targeted to any clonal frequency range of interest, we designed a 3-plate experiment to capture pairs with repertoire frequencies from 0.5% to less than 1 in 10 million. These experiments yielded over 300,000 confident TCR pairs with one 96-well plate alone providing over 200,000 pairs. This technology is not restricted by material; in a single experiment we used this method to identify 6,349 TCR pairs from tumor infiltrating lymphocytes in 9 tumor samples. Our TCR pairing method is highly scalable, and should be broadly applicable to all somatically rearranged immune receptor loci, including immunoglobulins.