Development and Analytical Validation of an Expanded Mutation Detection Panel for Next-Generation Sequencing of Thyroid Nodule Aspirates

Publication date: Available online 19 December 2019Source: The Journal of Molecular DiagnosticsAuthor(s): Kenny Kwabena Ablordeppey, Venkata Arun Timmaraju, Joanna Wanmin Song-Yang, Sharon Yaqoob, Christina Narick, Alidad Mireskandari, Sydney David Finkelstein, Gyanendra KumarABSTRACTMolecular analysis is used to evaluate the risk of malignancy for thyroid fine needle aspirates, identified as indeterminate by microscopic cytology, based on the detection of various oncogenic DNA mutations and fusion transcripts, or upon the use of various mRNAs or microRNA-based classifier algorithms. Our approach has been to use a combination test utilizing the detection of oncogenic mutations/ fusion transcripts and a micro-RNA 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 five oncogenes to 10 oncogenes and tumor suppressor genes and the detection of fusion transcripts was increased from six to 38. Here, 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 CAP/CLIA certified laboratory. Technical hurdles in NGS library preparation for the sequencing of both normal and high GC rich regions, and balanced amplification of various amplic...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research