Annotation of pseudogenous gene segments by high-throughput sequencing of rearranged lymphocyte receptor loci

The adaptive immune system generates a huge breadth of antigen-specific T (TCRs) and B cell receptors (BCRs) by combinatoric shuffling of gene segments. Lymphocytes undergo somatic recombination of variable (V), diversity (D), and joining (J) region gene segments, with deletions and non-templated insertions at the junctions, collectively encoding the CDR3 region. Many of the gene segments are annotated as non-functional; however, full annotation of genes as functional, pseudogene or ORF has proven elusive. We used high-throughput sequencing to identify 100K’s of rearranged TCRB and IGH receptors from a large cohort of healthy adults. We then annotated V and J gene segments as functional or pseudogenes based on selection. Random chance predicts that less than one-third of somatic rearrangements will lead to in-frame transcripts, but only lymphocytes with a productive receptor are selected. Non-functional rearrangements (out-of-frame or pseudogene) persist only as a second allele in mature T and B cells. Based on these data we annotated the functional status of each gene segment based on the realized ratio of productive to non-productive rearrangements. Some gene segments are currently mis-annotated: in TCRB 2 presumed-functional gene segments are pseudogenes, in IgH 3 presumed-functional genes are pseudogenes and 1 annotated pseudogene is functional. Additionally, in both loci we identified cases of gene segment functional/pseudogene status segregating among healthy subjects.