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Front Immunol. 2018 Feb 5;9:33. doi: 10.3389/fimmu.2018.00033. eCollection 2018.

Immune Repertoire Sequencing Using Molecular Identifiers Enables Accurate Clonality Discovery and Clone Size Quantification.

Author information

1
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States.
2
Department of Biomedical Engineering, Cockrell School of Engineering, The University of Texas at Austin, Austin, TX, United States.
3
McKetta Department of Chemical Engineering, Cockrell School of Engineering, The University of Texas at Austin, Austin, TX, United States.
4
ImmuDX, LLC, Austin, TX, United States.
5
School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.
6
Research Center of Special Environmental Biomechanics & Medical Engineering, Xi'an, Shaanxi, China.

Abstract

Unique molecular identifiers (MIDs) have been demonstrated to effectively improve immune repertoire sequencing (IR-seq) accuracy, especially to identify somatic hypermutations in antibody repertoire sequencing. However, evaluating the sensitivity to detect rare T cells and the degree of clonal expansion in IR-seq has been difficult due to the lack of knowledge of T cell receptor (TCR) RNA molecule copy number and a generalized approach to estimate T cell clone size from TCR RNA molecule quantification. This limited the application of TCR repertoire sequencing (TCR-seq) in clinical settings, such as detecting minimal residual disease in lymphoid malignancies after treatment, evaluating effectiveness of vaccination and assessing degree of infection. Here, we describe using an MID Clustering-based IR-Seq (MIDCIRS) method to quantitatively study TCR RNA molecule copy number and clonality in T cells. First, we demonstrated the necessity of performing MID sub-clustering to eliminate erroneous sequences. Further, we showed that MIDCIRS enables a sensitive detection of a single cell in as many as one million naïve T cells and an accurate estimation of the degree of T cell clonal expression. The demonstrated accuracy, sensitivity, and wide dynamic range of MIDCIRS TCR-seq provide foundations for future applications in both basic research and clinical settings.

KEYWORDS:

CMV-specific T cells; MID clustering-based IR-Seq TCR repertoire sequencing; molecular identifiers; naïve T cells; sub-clustering

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