Annotation of pseudogenous gene segments by massively parallel sequencing of rearranged lymphocyte receptor loci. R. O. Emerson1, A. M. Sherwood1, H. S. Robins2, C. S. Carlson2, M. J. Rieder1 1) Adaptive Biotechnologies, Seattle, WA; 2) Public Health Sciences Division, FHCRC, Seattle, WA.

   In order to generate a wide variety of functional T cell receptors and antibodies, lymphocytes undergo somatic rearrangement of the T cell receptor and immunoglobulin loci, each of which encodes dozens or hundreds of V, D and J gene segments in the germ line. As in many large gene families, many of these gene segments are classified as pseudogenes due to defects in primary sequence or motifs necessary for somatic rearrangement. Until now, a full annotation of pseudogene/functional status for each gene segment has proven elusive. Using next-gen sequencing of the T cell receptor beta (TCRB) and immunoglobulin heavy chain (IgH) loci in mature T and B cells from hundreds of healthy adults we have annotated the functional status of each V, D and J gene segment present in these loci and have identified functional genes that had been erroneously classified as pseudogenes. Briefly, random chance predicts that slightly less than one-third of somatic rearrangements at the TCRB and IgH loci will lead to in-frame transcripts with no premature stops; selection during lymphocyte maturation ensures that all mature T and B cells carry at least one rearrangement coding for a productive receptor, while the second allele also rearranges in some cells and can be out of frame, include a premature stop, or include a pseudogenous V, D or J gene segment. We have classified each gene segment as functional or pseudogene based on the proportion of in-frame rearrangements; in mature T and B cells the length of the CDR3 hypervariable region in rearrangements using functional gene segments has a pronounced periodicity at 3 nt, while no such feature exists in the case of pseudogenes. This discrepancy between functional and pseudogenous gene segments has allowed us to conclusively annotate the functional status of each gene segment in the complex TCRB and IgH immune receptor loci, and to examine the distribution of functional and pseudogenous alleles in hundreds of individuals.

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