title: A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains.
abstract: High-throughput sequencing of T cell receptor (TCR) genes is a powerful tool for analyses of antigen specificity, clonality and diversity of T lymphocytes. Here, we developed a new TCR repertoire analysis method using 454 DNA sequencing technology in combination with an adaptor-ligation mediated polymerase chain reaction (PCR). This method allows the amplification of all TCR genes without PCR bias. To compare gene usage, diversity and similarity of expressed TCR repertoires among individuals, we conducted next-generation sequencing (NGS) of TRA and TRB genes in peripheral blood mononuclear cells from 20 healthy human individuals.
  
  
  
  From a total of 267,037 sequence reads from 20 individuals, 149,216 unique sequence reads were identified. Preferential usage of several V and J genes were observed while some recombinations of TRAV with TRAJ appeared to be restricted. The extent of TCR diversity was not significantly different between TRA and TRB, while TRA repertoires were more similar between individuals than TRB repertoires were. The interindividual similarity of TRA depended largely on the frequent presence of shared TCRs among two or more individuals. A publicly available TRA had a near-germline TCR with a shorter CDR3. Notably, shared TRA sequences, especially those shared among a large number of individuals', often contained TCRα related with invariant TCRα derived from invariant natural killer T cells and mucosal-associated invariant T cells.
  
  
  
  These results suggest that retrieval of shared TCRs by NGS would be useful for the identification of potential new invariant TCRα chains. This NGS method will enable the comprehensive quantitative analysis of TCR repertoires at a clonal level.
authors.affiliation: Repertoire Genesis Incorporation, 104 Saito-Bioincubator, 7-7-15, Saito-asagi, Ibaraki, Osaka, 567-0085, Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Kitaura
authors.affiliation: Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, Sagamihara, Japan.|BITS. Co., Ltd, Tokyo, Japan.
authors.forename: Tadasu
authors.initials: T
authors.lastname: Shini
authors.affiliation: Repertoire Genesis Incorporation, 104 Saito-Bioincubator, 7-7-15, Saito-asagi, Ibaraki, Osaka, 567-0085, Japan. matsutani@repertoire.co.jp.
authors.forename: Takaji
authors.initials: T
authors.lastname: Matsutani
authors.affiliation: Repertoire Genesis Incorporation, 104 Saito-Bioincubator, 7-7-15, Saito-asagi, Ibaraki, Osaka, 567-0085, Japan.|Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, Sagamihara, Japan.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Suzuki
mesh_terms: D000328:Adult; D000368:Aged; D002999:Clone Cells; D019295:Computational Biology; D005260:Female; D014644:Genetic Variation; D064368:Healthy Volunteers; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000639:trends; D006801:Humans; D007564:Japan; D007963:Leukocytes, Mononuclear / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*
chemical_list: D016693:Receptors, Antigen, T-Cell, alpha-beta
keywords: Immune profiling; Invariant TCRα; Next generation sequencing; Repertoire; T cell receptor