Systematic comparative evaluation of methods for investigating the TCRβ repertoire

Xiao Liu; Wei Zhang; Xiaojing Zeng; Ruifang Zhang; Yuanping Du; Xueyu Hong; Hongzhi Cao; Zheng Su; Changxi Wang; Jinghua Wu; Chao Nie; Xun Xu; Karsten Kristiansen

doi:10.1371/journal.pone.0152464

Systematic comparative evaluation of methods for investigating the TCRβ repertoire

Xiao Liu, Wei Zhang, Xiaojing Zeng, Ruifang Zhang, Yuanping Du, Xueyu Hong, Hongzhi Cao, Zheng Su, Changxi Wang, Jinghua Wu, Chao Nie, Xun Xu, Karsten Kristiansen

22 Citations (Scopus)

77 Downloads (Pure)

Abstract

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.

Original language	English
Article number	e0152464
Journal	P L o S One
Volume	11
Issue number	3
Number of pages	18
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0152464
Publication status	Published - 2016

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Liu_2016_Systematic_comparative_evaluationFinal published version, 1.45 MBLicence: CC BY

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title = "Systematic comparative evaluation of methods for investigating the TCRβ repertoire",

abstract = "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.",

author = "Xiao Liu and Wei Zhang and Xiaojing Zeng and Ruifang Zhang and Yuanping Du and Xueyu Hong and Hongzhi Cao and Zheng Su and Changxi Wang and Jinghua Wu and Chao Nie and Xun Xu and Karsten Kristiansen",

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AU - Liu, Xiao

AU - Zhang, Wei

AU - Zeng, Xiaojing

AU - Zhang, Ruifang

AU - Du, Yuanping

AU - Hong, Xueyu

AU - Cao, Hongzhi

AU - Su, Zheng

AU - Wang, Changxi

AU - Wu, Jinghua

AU - Nie, Chao

AU - Xu, Xun

AU - Kristiansen, Karsten

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

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DO - 10.1371/journal.pone.0152464

M3 - Journal article

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AN - SCOPUS:84962426339

SN - 1932-6203

VL - 11

JO - PLoS Computational Biology

JF - PLoS Computational Biology

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ER -

Systematic comparative evaluation of methods for investigating the TCRβ repertoire

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