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

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 Graham, Jadee L. Neff, Kandelaria M. Rumilla, Umut Aypar, Benjamin R. Kipp, Robert Brian Jenkins, Jin Jen & 1 others Kevin C. Halling

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

We 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 the assay was tested by diluting RNA isolated from gene fusion–positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration–approved and investigational targeted therapies.

Original languageEnglish (US)
Pages (from-to)495-511
Number of pages17
JournalJournal of Molecular Diagnostics
Volume20
Issue number4
DOIs
StatePublished - Jul 1 2018

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RNA Sequence Analysis
Gene Fusion
Neoplasms
RNA
Investigational Therapies
Computational Biology
Fluorescence In Situ Hybridization
Genes
Complementary DNA
Food
Polymerase Chain Reaction
Messenger RNA
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

Cite this

Winters, J. L., Davila, J. I., McDonald, A. M., Nair, A. A., Fadra, N., Wehrs, R. N., ... Halling, K. C. (2018). Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors. Journal of Molecular Diagnostics, 20(4), 495-511. https://doi.org/10.1016/j.jmoldx.2018.03.007

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors. / Winters, Jennifer L.; Davila, Jaime I.; McDonald, Amber M.; Nair, Asha A.; Fadra, Numrah; Wehrs, Rebecca N.; Thomas, Brittany C.; Balcom, Jessica R.; Jin, Long; Wu, Xianglin; Voss, Jesse S.; Klee, Eric W; Oliver, Gavin R.; Graham, Rondell; Neff, Jadee L.; Rumilla, Kandelaria M.; Aypar, Umut; Kipp, Benjamin R.; Jenkins, Robert Brian; Jen, Jin; Halling, Kevin C.

In: Journal of Molecular Diagnostics, Vol. 20, No. 4, 01.07.2018, p. 495-511.

Research output: Contribution to journalArticle

Winters, JL, Davila, JI, McDonald, AM, Nair, AA, Fadra, N, Wehrs, RN, Thomas, BC, Balcom, JR, Jin, L, Wu, X, Voss, JS, Klee, EW, Oliver, GR, Graham, R, Neff, JL, Rumilla, KM, Aypar, U, Kipp, BR, Jenkins, RB, Jen, J & Halling, KC 2018, 'Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors', Journal of Molecular Diagnostics, vol. 20, no. 4, pp. 495-511. https://doi.org/10.1016/j.jmoldx.2018.03.007
Winters, Jennifer L. ; Davila, Jaime I. ; McDonald, Amber M. ; Nair, Asha A. ; Fadra, Numrah ; Wehrs, Rebecca N. ; Thomas, Brittany C. ; Balcom, Jessica R. ; Jin, Long ; Wu, Xianglin ; Voss, Jesse S. ; Klee, Eric W ; Oliver, Gavin R. ; Graham, Rondell ; Neff, Jadee L. ; Rumilla, Kandelaria M. ; Aypar, Umut ; Kipp, Benjamin R. ; Jenkins, Robert Brian ; Jen, Jin ; Halling, Kevin C. / Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors. In: Journal of Molecular Diagnostics. 2018 ; Vol. 20, No. 4. pp. 495-511.
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AU - Nair, Asha A.

AU - Fadra, Numrah

AU - Wehrs, Rebecca N.

AU - Thomas, Brittany C.

AU - Balcom, Jessica R.

AU - Jin, Long

AU - Wu, Xianglin

AU - Voss, Jesse S.

AU - Klee, Eric W

AU - Oliver, Gavin R.

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AU - Jenkins, Robert Brian

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AU - Halling, Kevin C.

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