title: Annotation of pseudogenic gene segments by massively parallel sequencing of rearranged lymphocyte receptor loci.
abstract: BACKGROUND
  The adaptive immune system generates a remarkable range of antigen-specific T-cell receptors (TCRs), allowing the recognition of a diverse set of antigens. Most of this diversity is encoded in the complementarity determining region 3 (CDR3) of the β chain of the αβ TCR, which is generated by somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) gene segments. Deletion and non-templated insertion of nucleotides at the D-J and V-DJ junctions further increases diversity. Many of these gene segments are annotated as non-functional owing to defects in their primary sequence, the absence of motifs necessary for rearrangement, or chromosomal locations outside the TCR locus.
  
  
  METHODS
  We sought to utilize a novel method, based on high-throughput sequencing of rearranged TCR genes in a large cohort of individuals, to evaluate the use of functional and non-functional alleles. We amplified and sequenced genomic DNA from the peripheral blood of 587 healthy volunteers using a multiplexed polymerase chain reaction assay that targets the variable region of the rearranged TCRβ locus, and we determined the presence and the proportion of productive rearrangements for each TCRβ V gene segment in each individual. We then used this information to annotate the functional status of TCRβ V gene segments in this cohort.
  
  
  RESULTS
  For most TCRβ V gene segments, our method agrees with previously reported functional annotations. However, we identified novel non-functional alleles for several gene segments, some of which were used exclusively in our cohort to the detriment of reported functional alleles. We also saw that some gene segments reported to have both functional and non-functional alleles consistently behaved in our cohort as either functional or non-functional, suggesting that some reported alleles were not present in the population studied.
  
  
  CONCLUSIONS
  In this proof-of-principle study, we used high-throughput sequencing of the TCRβ locus of a large cohort of healthy volunteers to evaluate the use of functional and non-functional alleles of individual TCRβ V gene segments. With some modifications, our method has the potential to be extended to gene segments in the α, γ, and δ TCR loci, as well as the genes encoding for B-cell receptor chains.
authors.affiliation: Adaptive Biotechnologies, Seattle, WA, USA. jdean@adaptivebiotech.com.
authors.forename: Jared
authors.initials: J
authors.lastname: Dean
authors.affiliation: Adaptive Biotechnologies, Seattle, WA, USA. remerson@adaptivebiotech.com.
authors.forename: Ryan O
authors.initials: RO
authors.lastname: Emerson
authors.affiliation: Adaptive Biotechnologies, Seattle, WA, USA. mvignali@adaptivebiotech.com.
authors.forename: Marissa
authors.initials: M
authors.lastname: Vignali
authors.affiliation: Adaptive Biotechnologies, Seattle, WA, USA. asherwood@adaptivebiotech.com.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Sherwood
authors.affiliation: Adaptive Biotechnologies, Seattle, WA, USA. mrieder@adaptivebiotech.com.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Rieder
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA, USA. ccarlson@fhcrc.org.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Carlson
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA, USA. hrobins@fhcrc.org.
authors.forename: Harlan S
authors.initials: HS
authors.lastname: Robins
mesh_terms: D000328:Adult; D000483:Alleles; D001483:Base Sequence; D022801:Complementarity Determining Regions / Q000235:genetics; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D056426:Genetic Loci; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D058977:Molecular Sequence Annotation; D016133:Polymerase Chain Reaction; D011544:Pseudogenes; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology / Q000502:physiology*
chemical_list: D022801:Complementarity Determining Regions; D016693:Receptors, Antigen, T-Cell, alpha-beta; D016692:Receptors, Antigen, T-Cell, gamma-delta