The use of non-functional clonotypes as a natural calibrator for quantitative bias correction in adaptive immune receptor repertoire profiling
- Skolkovo Institute of Science and Technology, Russian Federation
- Department of Genomics of Adaptive Immunity, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Federation
- Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Russian Federation
- Pirogov Russian National Research Medical University, Russian Federation
- Central European Institute of Technology, Masaryk University, Czech Republic
- Abu Dhabi Stem Cells Center, United Arab Emirates
Figures
Figure 1
Stability of TRBV and TRBJ genes frequencies calculated based on unique out-of-frame rearrangements after Yellow fever vaccination (model of acute viral infection).
Out-of-frame clonotypes for frequencies calculation were extracted from low-biased 5’ Rapid Amplification of cDNA Ends (RACE) TRB repertoires of PBMC (peripheral blood mononuclear cells) samples …
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Figure 1—source data 1
XLSX table.
TRB V and J frequencies for plots in Figure 1.
- https://cdn.elifesciences.org/articles/69157/elife-69157-fig1-data1-v3.xlsx
Figure 2 with 1 supplement
Dependence of the Over Amplification Rate on the TCR repertoire profiling method.
(a) Comparison of Over Amplification Rate (OAR) values variances for TRB repertoires obtained with 5’-Rapid Amplification of cDNA Ends (RACE), one-side multiplex (VMPlex), and two-side multiplex …
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Figure 2—source data 1
XLSX table.
Over Amplification Rate (OAR) values used for the plots in Figure 2.
- https://cdn.elifesciences.org/articles/69157/elife-69157-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Comparison of population V/J segment frequencies with individual frequencies obtained using different TCR repertoire profiling methods.
V- and J-gene frequencies among unique out-of-frame rearrangements calculated using biased (two-side multiplex, empty circles) and low-biased (Rapid Amplification of cDNA Ends [RACE], filled …
Figure 3
Over Amplification Rate of low-biased TCR and BCR repertoires in different lymphocyte subsets.
(a) Distribution of Over Amplification Rates of V- and J-genes in Rapid Amplification of cDNA Ends (RACE)-based repertoires of TCR and BCR (the empty dots represent average OARs among TCR …
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Figure 3—source data 1
XLSX table.
Over Amplification Rate (OAR) values used for the plots in Figure 3.
- https://cdn.elifesciences.org/articles/69157/elife-69157-fig3-data1-v3.xlsx
Figure 4
Factors impacting Over Amplification Rate (OAR) calculation accuracy.
(a) Impact of highly proliferated top non-functional clonotype on OAR calculation accuracy in low-biased Rapid Amplification of cDNA Ends (RACE)-based TRB repertoire (Data: SRR19594184). (b) Impact …
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Figure 4—source data 1
XLSX table.
Over Amplification Rate (OAR) values used for the plots in Figure 4.
- https://cdn.elifesciences.org/articles/69157/elife-69157-fig4-data1-v3.xlsx
Figure 5
Implementation of OAR-based correction of quantitative bias in TCR repertoire.
(a) Flowchart of iROAR (immune Repertoire Over Amplification Removal) algorithm. UCF – Frequency calculated using unique clones counts (denominator of Over Amplification Rate [OAR]), pUCF – …
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Figure 5—source data 1
XLSX table.
Clonal frequencies used for the plots in Figure 5.
- https://cdn.elifesciences.org/articles/69157/elife-69157-fig5-data1-v3.xlsx
Figure 6
Effect of iROAR (immune Repertoire Over Amplification Removal)-based PCR bias correction in MPlex repertoire on similarity with low-biased Rapid Amplification of cDNA Ends (RACE)-based repertoire obtained from the same RNA sample.
(a) Pearson correlation coefficient; (b) R2 measure; (c) Morisita-Horn similarity index. **** p<0.0001 (two-tailed Wilcoxon matched-pairs signed rank test, CI = 0.95). Dataset: PRJNA548335 three …
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Figure 6—source data 1
XLSX table.
Pearson R, R2, and Morisita-Horn index values used for the plots in Figure 6.
- https://cdn.elifesciences.org/articles/69157/elife-69157-fig6-data1-v3.xlsx
Figure 7
Comparison of Over Amplification Rate (OAR)-based and biological spike-in-based approaches for multiplex PCR bias detection.
Pearson’s correlation coefficient for V-segments bias measure (a) and J-segments bias measure (b). Column and row titles: PM = Primer mix, R=replica. (c) Correlation of VJ combination bias …
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Figure 7—source data 1
XLSX table.
iROAR (immune Repertoire Over Amplification Removal)-based and spike-in-based PCR bias measures used for the plots in Figure 7.
- https://cdn.elifesciences.org/articles/69157/elife-69157-fig7-data1-v3.xlsx
Figure 8
Convergence of Over Amplification Rate (OAR), clonotype, and V/J frequencies between two TRA repertoire before and after iROAR (immune Repertoire Over Amplification Removal) based bias correction.
(a) OAR values changes in four test TRA libraries after PCR bias correction using iROAR. (b) TRAV and TRAJ frequency changes after PCR bias correction using iROAR (Sample: Primer mix 1 Replica 1). (c…
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Figure 8—source data 1
XLSX table.
Clonal frequencies, TRB V and J frequencies and Over Amplification Rate (OAR) values used for the plots in Figure 8.
- https://cdn.elifesciences.org/articles/69157/elife-69157-fig8-data1-v3.xlsx
Tables
Table 1
The number of spike-in and target clonotypes in test TRA libraries.
| Sample | Spike-in clonotypes | Target clonotypes | ||||
|---|---|---|---|---|---|---|
| Number | Read count | Coverage | Number | Read count | Coverage | |
| Primer mix 1 Replica 1 | 3571 | 30,474 | 8.53 | 39,911 | 348,792 | 8.74 |
| Primer mix 1 Replica 2 | 2698 | 19,420 | 7.20 | 35,818 | 303,494 | 8.47 |
| Primer mix 2 Replica 1 | 3439 | 34,717 | 10.10 | 40,209 | 425,508 | 10.58 |
| Primer mix 2 Replica 2 | 2298 | 24,823 | 10.80 | 33,406 | 383,615 | 11.48 |
Additional files
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Supplementary file 1
XLSX table.
Dataset accession numbers and references to the original studies generated it.
- https://cdn.elifesciences.org/articles/69157/elife-69157-supp1-v3.xlsx
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Supplementary file 2
XLSX table.
Comparison of iROAR (immune Repertoire Over Amplification Removal) algorithm with the existing approaches for PCR bias removal in human adaptive immune receptor repertoires.
- https://cdn.elifesciences.org/articles/69157/elife-69157-supp2-v3.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69157/elife-69157-transrepform1-v3.docx
