Analyzing T cell repertoire diversity by high-throughput sequencing

Diversity on a large scale is one of the most striking and powerful features utilized by the mammalian immune system to fight off a vast universe of pathogens. The T-cell driven immune response is characterized by a multitude of distinct receptors capable of antigen recognition with high specificity. Using high-throughput sequencing we are able to investigate the T cell receptor (TCR) repertoire as the collection of its individual receptors, aiming to profile the global properties of the complementarily determining region 3 (CDR3) of the TCR in human immunity. However, analysis of the data is highly sensitive to single nucleotide polymoprhisms, read length and error rate, accuracy in mapping to a genomic reference, and our ability to translate the sequence, in silico, in the appropriate reading frame. We have developed a computational pipeline that performs error correction on overlapping paired-end long (250 nt) reads, and maps the reads unambiguously to V and J cassettes corresponding to TCR-α and -β chains. Our methods were applied to functional T cell receptors from healthy blood tissue and to several patients with low grade glioma (LGG) and glioblastoma multiforme (GBM). We used Shannon entropy to measure levels of diversity in the productive T cell repertoire and observed that greater than 50% of the TCR diversity can be explained by V, J cassette usage.