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Validation of a Next-Generation Sequencing-based T-Cell Receptor Gamma Gene Rearrangement Diagnostic Assay: Transitioning from Capillary Electrophoresis to Next-Generation Sequencing.
Validation of a Next-Generation Sequencing-based T-Cell Receptor Gamma Gene Rearrangement Diagnostic Assay: Transitioning from Capillary Electrophoresis to Next-Generation Sequencing. The Journal of molecular diagnostics : JMD Ho, C. C., Tung, J. K., Zehnder, J. L., Zhang, B. M. 2021Abstract
Assessment of T-cell receptor gamma (TRG) gene rearrangement is an important consideration in the diagnostic workup of lymphoproliferative diseases. Although fragment analysis by PCR and capillary electrophoresis (CE) is the current standard for such assessment in clinical molecular diagnostic laboratories, it does not provide sequence information and is only semi-quantitative. Next-generation sequencing (NGS)-based assays are an attractive alternative to the conventional fragment-size based methods since they generate results with specific clonotype sequence information and allow for more accurate quantitation. We therefore evaluated various test parameters and performance characteristics for a commercially available NGS-based TRG gene rearrangement assay by testing 101 clinical samples previously characterized by fragment analysis. The NGS TRG assay showed an overall accuracy of 83% and an analytical specificity of 100%, as compared to the CE-based assay. The concordance rate was 88~95% for V?1-8, V?10 and V?11 gene families, but lower for the V?9 gene family. This difference was mostly attributed to the incomplete polyclonal symmetry resulting from the two-tube CE assay versus the one-tube design of the NGS assay. The NGS assay also demonstrated strengths in distinguishing different clonotypes with the same fragment size. Our clinical validation demonstrated robust performance of the NGS-based TRG assay and identified potential pitfalls associated with CE assay design that are important for understanding the observed discrepancies with the CE-based assay.
View details for DOI 10.1016/j.jmoldx.2021.03.008
View details for PubMedID 33892183