title: Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood.
abstract: BACKGROUND
  As one of the genetic mechanisms for adaptive immunity, V(D)J recombination generates an enormous repertoire of T-cell receptors (TCRs). With the development of high-throughput sequencing techniques, systematic exploration of V(D)J recombination becomes possible. Multiplex PCR has been previously developed to assay immune repertoire; however, the use of primer pools leads to inherent biases in target amplification. In our study, we developed a "single-primer" ligation-anchored PCR method that may amplify the repertoire with much less biases.
  
  
  RESULTS
  By utilizing a universal primer paired with a single primer targeting the conserved constant region, we amplified TCR-beta (TRB) variable regions from total RNA extracted from blood. Next-generation sequencing libraries were then prepared for Illumina HiSeq 2500 sequencer, which generates 151-bp read length to cover the entire V(D)J recombination region. We evaluated this approach on blood samples from healthy donors and from patients with malignant and benign meningiomas. Mapping of sequencing data showed that 64% to 96% of mapped TCRV-containing reads belong to TRB subtype. An increased usage of specific V segments and V-J pairing were observed in malignant meningiomas samples. The CDR3 sequences of the expanded V-J pairs were distinct in each malignant individual, even for pairing of TRBV7-3 with TRBJ2-2 that showed increased usage in both cases.
  
  
  CONCLUSIONS
  We demonstrated the technical feasibility and effectiveness of ligation-anchored PCR approach in capturing the TCR-beta landscapes. Further development of this technology may enable a comprehensive delineation of immune repertoire, including other forms of TCRs as well as immunoglobulins.
authors.affiliation: Zilkha Neurogenetic Institute, University of Southern California, 1501 San Pablo Street, Los Angeles, CA, 90089, USA. fangao@mit.edu.|Current address: The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Boston, MA, 02139, USA. fangao@mit.edu.
authors.forename: Fan
authors.initials: F
authors.lastname: Gao
authors.affiliation: Zilkha Neurogenetic Institute, University of Southern California, 1501 San Pablo Street, Los Angeles, CA, 90089, USA. kaiwang@usc.edu.|Department of Psychiatry, University of Southern California, Los Angeles, CA, 90089, USA. kaiwang@usc.edu.
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D008297:Male; D008579:Meningioma / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000097:blood / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D016693:Receptors, Antigen, T-Cell, alpha-beta; D012313:RNA