High throughput sequencing of T-cell receptor repertoire using dry blood spots

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...

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Main Authors: Shang-Gin Wu, Wenjing Pan, Hongna Liu, Miranda L. Byrne-Steele, Brittany Brown, Mollye Depinet, Xiaohong Hou, Jian Han, Song Li
Format: Article
Language:English
Published: BMC 2019-02-01
Series:Journal of Translational Medicine
Subjects:
Online Access:http://link.springer.com/article/10.1186/s12967-019-1796-4
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spelling doaj-886eb718cb1c4dacab0d1663fd0e2f352020-11-25T03:37:04ZengBMCJournal of Translational Medicine1479-58762019-02-0117111210.1186/s12967-019-1796-4High throughput sequencing of T-cell receptor repertoire using dry blood spotsShang-Gin Wu0Wenjing Pan1Hongna Liu2Miranda L. Byrne-Steele3Brittany Brown4Mollye Depinet5Xiaohong Hou6Jian Han7Song Li8Department of Internal Medicine, National Taiwan University Hospital, National Taiwan UniversityHudsonAlpha Institute for BiotechnologyiCubate Inc.iRepertoire Inc.iRepertoire Inc.iRepertoire Inc.iRepertoire Inc.HudsonAlpha Institute for BiotechnologyiRepertoire Inc.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.http://link.springer.com/article/10.1186/s12967-019-1796-4T cell receptorTCR repertoireCDR3Dry blood spotsNext-generation sequenceDiversity
collection DOAJ
language English
format Article
sources DOAJ
author Shang-Gin Wu
Wenjing Pan
Hongna Liu
Miranda L. Byrne-Steele
Brittany Brown
Mollye Depinet
Xiaohong Hou
Jian Han
Song Li
spellingShingle Shang-Gin Wu
Wenjing Pan
Hongna Liu
Miranda L. Byrne-Steele
Brittany Brown
Mollye Depinet
Xiaohong Hou
Jian Han
Song Li
High throughput sequencing of T-cell receptor repertoire using dry blood spots
Journal of Translational Medicine
T cell receptor
TCR repertoire
CDR3
Dry blood spots
Next-generation sequence
Diversity
author_facet Shang-Gin Wu
Wenjing Pan
Hongna Liu
Miranda L. Byrne-Steele
Brittany Brown
Mollye Depinet
Xiaohong Hou
Jian Han
Song Li
author_sort Shang-Gin Wu
title High throughput sequencing of T-cell receptor repertoire using dry blood spots
title_short High throughput sequencing of T-cell receptor repertoire using dry blood spots
title_full High throughput sequencing of T-cell receptor repertoire using dry blood spots
title_fullStr High throughput sequencing of T-cell receptor repertoire using dry blood spots
title_full_unstemmed High throughput sequencing of T-cell receptor repertoire using dry blood spots
title_sort high throughput sequencing of t-cell receptor repertoire using dry blood spots
publisher BMC
series Journal of Translational Medicine
issn 1479-5876
publishDate 2019-02-01
description 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.
topic T cell receptor
TCR repertoire
CDR3
Dry blood spots
Next-generation sequence
Diversity
url http://link.springer.com/article/10.1186/s12967-019-1796-4
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