Design, optimization, and multisite evaluation of a targeted next-generation sequencing assay system for RNA fusions and exon-skipping events in non-small cell lung cancer.

Blidner, R., Haynes, B. C., Schmitt, S., Pessetto, Z. Y., Godwin, A. K., Su, D., Hurban, P., van Kempen, L., Aguirre, M. L., Gokul, S., Cardwell, R. D. & Latham, G. J., 1-Mar-2019, In : Journal of molecular diagnostics. 21, 2, p. 352-365 14 p.

Research output: Contribution to journalArticleAcademicpeer-review

  • R Blidner
  • B.C. Haynes
  • S Schmitt
  • Z.Y. Pessetto
  • A.K. Godwin
  • D Su
  • P Hurban
  • Leon van Kempen
  • Maria Leonor Aguirre
  • S Gokul
  • R D Cardwell
  • G J Latham
Lung cancer accounts for approximately 14% of all newly diagnosed cancers and is the leading cause of cancer-related deaths. Chimeric RNA resulting from gene fusions (RNA fusions) and other RNA splicing errors are driver events and clinically addressable targets for non-small cell lung cancer (NSCLC). The reliable assessment of these RNA markers by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We describe the development and verification of a system for targeted RNA sequencing 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 attributable to index hopping events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multiphase study conducted at five independent laboratories. The results show 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 sequencing of formalin-fixed, paraffin-embedded samples relevant to NSCLC.
Original languageEnglish
Pages (from-to)352-365
Number of pages14
JournalJournal of molecular diagnostics
Issue number2
Publication statusPublished - 1-Mar-2019

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