Validated next generation sequencing (NGS) assay for the characterization of the T-cell repertoire from RNA

GCP validated NGS assay

Activation of T-cells during cell-mediated immunity is initiated by the stimulation of the T- cell receptor (TCR) by major histocompatibility complex-antigen complexes. While the entire TCR chain is diverse, most of the diversity is concentrated in a hypervariable complementarity-determining region 3 (CDR3) loop, the center of the antigen-binding site for the TCR.  The frequency of a specific CDR3 sequence within the T-cell repertoire is a surrogate for the abundance of its corresponding T-cell clone. Deep sequencing of the TCR CDR3 region assists with resolving T-cell diversity, and, in oncology, detects specific clones or changes in clonality associated with anti-tumor immune responses.  Here, we have validated an RNA based TCRβ/γ next-generation sequencing assay for use with whole blood, peripheral blood mononuclear cells (PBMCs) and formalin-fixed paraffin-embedded (FFPE) tissues.

In this study, TCR analysis was performed using RNA or total nucleic acids derived from whole blood, PBMCs or tumor FFPE specimens.  The TCRβ/γ sequencing assay entailed gene specific cDNA synthesis, preparation of sequencing libraries, TCR gene specific amplification, sample barcoding and sequencing.  A 2x150 bp sequencing was performed and ≥2 million paired-end reads for each sample were obtained.  Data analysis was performed using Archer Analysis software.

Download our poster from SITC 2018 to learn more about our validated RNA-based NGS assay for the analysis of TCRβ/γ in whole blood, PBMC and FFPE specimens.

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