Abstract
BackgroundImmunology 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.MethodsFinger-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).ResultsWe 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.ConclusionsThe 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.
Highlights
Immunology research, 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
We have demonstrated that storage of dry blood spots (DBS) in filter cards up to 28 days at either room temperature or − 20 °C has no effect on unique CDR3 discovery
After cDNA pooling of the TCRβ library and quantification, next generation sequencing (NGS) sequencing was performed, and the data was analyzed as described previously
Summary
Immunology research, 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. Immunology research, 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 [1]. TCR repertoire sequencing is widely used to evaluate the immune system [2]. TCR repertoires of patients were explored in a variety of disorders—patients under cancer immunotherapy, autoimmune disease [rheumatoid arthritis (RA), ankylosing spondylitis], or subject to virus infection (hepatitis, human immunodeficiency virus (HIV)) [3,4,5,6,7]. The costs, participant burden, regulatory constraints, and logistics associated with venipuncture and RNA handling are major barriers to clinical application or community-based research on various diseases
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