Systematic Comparative Evaluation of Methods for Investigating the TCRβ Repertoire

PLoS One. 2016 Mar 28;11(3):e0152464. doi: 10.1371/journal.pone.0152464. eCollection 2016.


High-throughput sequencing has recently been applied to profile the high diversity of antibodyome/B cell receptors (BCRs) and T cell receptors (TCRs) among immune cells. To date, Multiplex PCR (MPCR) and 5'RACE are predominately used to enrich rearranged BCRs and TCRs. Both approaches have advantages and disadvantages; however, a systematic evaluation and direct comparison of them would benefit researchers in the selection of the most suitable method. In this study, we used both pooled control plasmids and spiked-in cells to benchmark the MPCR bias. RNA from three healthy donors was subsequently processed with the two methods to perform a comparative evaluation of the TCR β chain sequences. Both approaches demonstrated high reproducibility (R2 = 0.9958 and 0.9878, respectively). No differences in gene usage were identified for most V/J genes (>60%), and an average of 52.03% of the CDR3 amino acid sequences overlapped. MPCR exhibited a certain degree of bias, in which the usage of several genes deviated from 5'RACE, and some V-J pairings were lost. In contrast, there was a smaller rate of effective data from 5'RACE (11.25% less compared with MPCR). Nevertheless, the methodological variability was smaller compared with the biological variability. Through direct comparison, these findings provide novel insights into the two experimental methods, which will prove to be valuable in immune repertoire research and its interpretation.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Complementarity Determining Regions / genetics
  • Female
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Male
  • Multiplex Polymerase Chain Reaction*
  • Nucleic Acid Amplification Techniques*
  • RNA / chemistry
  • RNA / isolation & purification
  • RNA / metabolism*
  • Receptors, Antigen, T-Cell, alpha-beta / genetics*
  • Receptors, Antigen, T-Cell, alpha-beta / metabolism
  • Reproducibility of Results
  • Sequence Analysis, RNA


  • Complementarity Determining Regions
  • Receptors, Antigen, T-Cell, alpha-beta
  • RNA

Grant support

This paper was funded by Beijing Genomics Institute (BGI). The funder provided support in the form of salaries for authors XL, WZ, XJZ, XH, RZ, YD, CW, HC, ZS, JW, CN, XX, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.