Development and Analytical Validation of an Expanded Mutation Detection Panel for Next-Generation Sequencing of...

Molecular analysis is used to evaluate the risk of malignancy for thyroid fine-needle aspirates, identified as indeterminate by microscopic cytology, on the basis of the detection of various oncogenic DNA mutations and fusion transcripts or on the use of various mRNAs or miRNA-based classifier algorithms. Our approach has been to use a combination test using the detection of oncogenic mutations/fusion transcripts and an miRNA expression-based classifier algorithm. To improve the performance of the combination test, the next-generation sequencing (NGS)-based mutational panel was expanded from the detection of 5 oncogenes to 10 oncogenes and tumor suppressor genes and the detection of fusion transcripts was increased from 6 to 38. Herein, we describe the assay development of the expanded panel NGS test and optimization of various steps for the library preparation of multiplexed target genes to maintain quality parameters for sequencing and to improve the robustness of the test for use in clinical testing in a College of American Pathologists/Clinical Laboratory Improvements Amendments-certified laboratory. Technical hurdles in NGS library preparation for the sequencing of both normal and high guanine-cytosine-rich regions, and balanced amplification of various amplicons in highly multiplexed PCRs, were successfully overcome. Analytical validation as a laboratory-developed test (ThyGeNEXT) included the demonstration of assay reproducibility, lower limit of detection, as well as other fundamental quality parameters.