domingo, 13 de noviembre de 2016

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

2016 Nov 1. pii: S1525-1578(16)30182-9. doi: 10.1016/j.jmoldx.2016.07.009. [Epub ahead of print]

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

Xu L1, You X1, Zheng P2, Zhang BM1, Gupta PK2, Lavori P3, Meyer E2, Zehnder JL4.

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Abstract

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.