Journal of Translational Medicine
High throughput sequencing of T-cell receptor repertoire using dry blood spots
- Shang-Gin Wu†,
- Wenjing Pan†,
- Hongna Liu,
- Miranda L. Byrne-Steele,
- Brittany Brown,
- Mollye Depinet,
- Xiaohong Hou,
- Jian Han and
- Song Li
†Contributed equally
Journal of Translational Medicine201917:47
© The Author(s) 2019
- Received: 8 January 2019
- Accepted: 9 February 2019
- Published: 18 February 2019
Abstract
Background
Immunology research, particularly next generation sequencing (NGS) of the immune T-cell receptor β (TCRβ) repertoire, has advanced progression in several fields, including treatment of various cancers and autoimmune diseases. This study aimed to identify the TCR repertoires from dry blood spots (DBS), a method that will help collecting real-world data for biomarker applications.
Methods
Finger-prick blood was collected onto a Whatman filter card. RNA was extracted from DBS of the filter card, and fully automated multiplex PCR was performed to generate a TCRβ chain library for next generation sequencing (NGS) analysis of unique CDR3s (uCDR3).
Results
We demonstrated that the dominant clonotypes from the DBS results recapitulated those found in whole blood. According to the statistical analysis and laboratory confirmation, 40 of 2-mm punch disks from the filter cards were enough to detect the shared top clones and have strong correlation in the uCDR3 discovery with whole blood. uCDR3 discovery was neither affected by storage temperatures (room temperature versus − 20 °C) nor storage durations (1, 14, and 28 days) when compared to whole blood. About 74–90% of top 50 uCDR3 clones of whole blood could also be detected from DBS. A low rate of clonotype sharing, 0.03–1.5%, was found among different individuals.
Conclusions
The DBS-based TCR repertoire profiling method is minimally invasive, provides convenient sampling, and incorporates fully automated library preparation. The system is sensitive to low RNA input, and the results are highly correlated with whole blood uCDR3 discovery allowing study scale-up to better understand the relationship and mutual influences between the immune and diseases.
Keywords
- T cell receptor
- TCR repertoire
- CDR3
- Dry blood spots
- Next-generation sequence
- Diversity
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