Systematic Comparative Evaluation of Methods for Investigating the TCRβ Repertoire.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

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

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.

Blood. 2009 Nov 5;114(19):4099-107. (PMID: 19706884)
Genome Res. 2009 Oct;19(10):1817-24. (PMID: 19541912)
Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1518-23. (PMID: 20080641)
Sci Transl Med. 2009 Dec 23;1(12):12ra23. (PMID: 20161664)
Immunol Lett. 2010 Sep 6;133(1):42-8. (PMID: 20621124)
Sci Transl Med. 2010 Sep 1;2(47):47ra64. (PMID: 20811043)
MAbs. 2011 Jan-Feb;3(1):17-20. (PMID: 21099370)
J Immunol. 2011 Apr 1;186(7):4285-94. (PMID: 21383244)
Genome Res. 2011 May;21(5):790-7. (PMID: 21349924)
Oncotarget. 2011 Mar;2(3):178-85. (PMID: 21399237)
J Immunol Methods. 2012 Jan 31;375(1-2):14-9. (PMID: 21945395)
Immunology. 2012 Mar;135(3):183-91. (PMID: 22043864)
Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):13463-8. (PMID: 23898164)
Nat Commun. 2013;4:2333. (PMID: 23995877)
Curr Opin Immunol. 2013 Oct;25(5):646-52. (PMID: 24140071)
Genetics. 2015 Oct;201(2):459-72. (PMID: 26297338)
J Comput Biol. 2000 Feb-Apr;7(1-2):203-14. (PMID: 10890397)
Bioinformatics. 2004 Aug 4;20 Suppl 1:i379-85. (PMID: 15262823)
Biometrics. 1987 Dec;43(4):783-91. (PMID: 3427163)
J Mol Biol. 1990 Oct 5;215(3):403-10. (PMID: 2231712)
Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W6-9. (PMID: 16845079)
J Immunol Methods. 2007 Apr 10;321(1-2):182-95. (PMID: 17337271)
Nat Rev Immunol. 2008 Mar;8(3):231-8. (PMID: 18301425)
Science. 2009 May 8;324(5928):807-10. (PMID: 19423829)
Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20216-21. (PMID: 19875695)

0 (Complementarity Determining Regions)
0 (Receptors, Antigen, T-Cell, alpha-beta)
63231-63-0 (RNA)

Date Created: 20160329 Date Completed: 20160805 Latest Revision: 20191210

20231215

PMC4809601

10.1371/journal.pone.0152464

27019362