Methodologic Considerations in the Application of Next-Generation Sequencing of Human TRB Repertoires for Clinical Use

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• 作者：Liwen Xu^&lowast ; ; Xiaoqing You^&lowast ; ; PingPing Zheng^&dagger ; ; Bing M. Zhang^&lowast ; ; Puja K. Gupta^&dagger ; ; Philip Lavori^&Dagger ; ; Everett Meyer^&dagger ; ; James L. Zehnder^&lowast ; ; ^{zehnder@stanford.edu}
• 刊名：The Journal of Molecular Diagnostics
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：19
• 期：1
• 页码：72-83
• 全文大小：2072 K
• 卷排序：19

文摘

Next-generation sequencing (NGS) of immune receptors has become a standard tool to assess minimal residual disease (MRD) in patients treated for lymphoid malignancy, and it is being used to study the T-cell repertoire in many clinical settings. To better understanding the potential clinical utility and limitations of this application outside of MRD, we developed a BIOMED-2 primer–based NGS method and characterized its performance in controls and patients with graft-versus-host disease (GVHD) after allogeneic hematopoietic transplant. For controls and patients with GVHD, replicate sequencing of the same T-cell receptor β (TRB) libraries was highly reproducible. Higher variability was observed in sequencing of different TRB libraries made from the same DNA stock. Variability was increased in patients with GVHD compared with controls; patients with GVHD also had lower diversity than controls. In the T-cell repertoire of a healthy person, approximately 99.6% of the CDR3 clones were in low abundance, with frequency <10−3. A single library could identify >93% of the clones with frequency ≥10−3 in the repertoire. Sequencing in duplicate increased the average detection rate to >97%. This work demonstrates that NGS reliably and robustly characterizes TRB populations in healthy individuals and patients with GVHD with frequency ≥10−3 and provides a methodologic framework for applying NGS immune repertoire methods to clinical testing applications beyond MRD.