Methodologic Considerations in the Application of Next-Generation Sequencing of Human TRB Repertoires for Clinical Use.
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 ≥10-3in 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 ≥10-3and provides a methodologic framework for applying NGS immune repertoire methods to clinical testing applications beyond MRD.
Department of Pathology, Stanford School of Medicine, Stanford University, Stanford, California.
Liwen
L
Xu
Department of Pathology, Stanford School of Medicine, Stanford University, Stanford, California.
Xiaoqing
X
You
Division of Blood and Marrow Transplantation, Department of Medicine, Stanford School of Medicine, Stanford University, Stanford, California.
PingPing
P
Zheng
Department of Pathology, Stanford School of Medicine, Stanford University, Stanford, California.
Bing M
BM
Zhang
Division of Blood and Marrow Transplantation, Department of Medicine, Stanford School of Medicine, Stanford University, Stanford, California.
Puja K
PK
Gupta
Department of Biomedical Data Science, Stanford School of Medicine, Stanford University, Stanford, California.
Philip
P
Lavori
Division of Blood and Marrow Transplantation, Department of Medicine, Stanford School of Medicine, Stanford University, Stanford, California.
Everett
E
Meyer
Department of Pathology, Stanford School of Medicine, Stanford University, Stanford, California. Electronic address: zehnder@stanford.edu.
James L
JL
Zehnder
D001483:Base Sequence; D016022:Case-Control Studies; D005787:Gene Frequency; D014644:Genetic Variation; D006086:Graft vs Host Disease / Q000175:diagnosis* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D018365:Neoplasm, Residual; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014184:Transplantation, Homologous
D016693:Receptors, Antigen, T-Cell, alpha-beta