Design, optimization, and multisite evaluation of a targeted next-generation sequencing assay system for chimeric RNAs from gene fusions and exon-skipping events in non–small-cell lung cancer

We describe the development and verification of a system for targeted RNA-Seq for the analysis of challenging, low-input solid tumor biopsies that includes reagents for nucleic acid quantification and library preparation, run controls, and companion bioinformatics software. Assay development reconciled sequence discrepancies in public databases, created predictive formalin-fixed, paraffin-embedded RNA qualification metrics, and eliminated read misidentification due to “index hopping” events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multi-phase study conducted at five independent laboratories. The results demonstrate accurate, reproducible, and sensitive detection of RNA fusions, alternative splicing events, and other expression markers of NSCLC. This comprehensive approach, combining sample quantification, quality control, library preparation, and interpretive bioinformatics software, may accelerate the routine implementation of targeted RNA-Seq of formalin-fixed, paraffin-embedded samples relevant to NSCLC.
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research