Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors

Publication date: July 2018Source: The Journal of Molecular Diagnostics, Volume 20, Issue 4Author(s): Jennifer L. Winters, Jaime I. Davila, Amber M. McDonald, Asha A. Nair, Numrah Fadra, Rebecca N. Wehrs, Brittany C. Thomas, Jessica R. Balcom, Long Jin, Xianglin Wu, Jesse S. Voss, Eric W. Klee, Gavin R. Oliver, Rondell P. Graham, Jadee L. Neff, Kandelaria M. Rumilla, Umut Aypar, Benjamin R. Kipp, Robert B. Jenkins, Jin JenWe assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of t...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research