An Efficient Method for Identifying Gene Fusions by Targeted
Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.
Citation: Scolnick JA, Dimon M, Wang I-C, Huelga SC, Amorese DA (2015) An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples. PLoS ONE 10(7): e0128916. https://doi.org/10.1371/journal.pone.0128916
Editor: Keping Xie, The University of Texas MD Anderson Cancer Center, UNITED STATES
Received: January 5, 2015; Accepted: May 1, 2015; Published: July 1, 2015
Copyright: © 2015 Scolnick et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All sequencing files are available from the SRA database (accession number SRP051118).
Funding: NuGEN Technologies provided funding for this work. The funder provided support in the form of salaries for all authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
Competing interests: JS, IW, SH and DA are current employees of NuGEN Technologies and MD was a paid consultant to NuGEN Technologies. NuGEN Technologies also provided the funding for this research and markets the Ovation Target Enrichment Fusion Panel described in the manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
Introduction
Cancer cells frequently contain chromosomal rearrangements that lead to the formation of fusion genes expressed in the cell []. These fusion genes can act as drivers for cell growth. For example, the Philadelphia Chromosome rearrangement, originally identified in chronic myelogenous leukemia [] (CML), is the result of a translocation between chromosomes 9 and 22 which leads to the expression of a fusion gene combining the BCR and ABL kinases []. While the BCR-ABL fusion gene leads to uncontrolled cell growth, when the fusion gene is identified in a patient, CML can be treated successfully with tyrosine kinase inhibitors.
Gene fusions are typically identified in cells by fluorescent in situ hybridization (FISH), a technique in which selected regions of chromosomes are fluorescently labeled through the hybridization of specific oligonucleotide probes. Aclinician must then visually identify two chromosomal regions that have rearranged in a known pattern. FISH has many problems as a technique including being a difficult, low throughput procedure, required knowledge of both gene fusion partners, low spatial resolution, the availability of the necessary fluorescent probes and the need for highly trained personnel to decide if a fusion event has taken place []. These difficulties apply to both clinical and research laboratories, thus limiting the potential understanding of gene fusion events in human biology.
One recent alternative to FISH when studying gene fusions is high throughput sequencing. In particular, RNA sequencing (RNA-Seq) has been used to identify gene fusions that are transcribed into RNAs within various cells []. Software has been specifically developed to identify gene fusion events in RNA-Seq data [] and many previously unknown gene fusions have been identified this way. However, while RNA-Seq is a powerful tool for identifying gene fusion transcripts, it is currently cost prohibitive to sequence RNA from a tumor sample, in which only a small fraction of the total cells are expressing the gene fusion, and obtain enough sequencing reads to identify those fusion events. Here we describe an innovative new assay for gene fusion discovery and verification. Based on Single Primer Enrichment Technology (SPET, ), we target particular RNAs for sequencing, thus reducing the number of uninformative sequencing reads and increasing the sensitivity of gene fusion detection compared to standard RNA-Seq methods. The SPET based assay is easy to use, has low RNA input requirements and can be used with RNA from formalin fixed, paraffin embedded (FFPE) tissue, which is important for clinically relevant samples. In addition, the assay is fully customizable to target any gene or set of genes in any genome. To show the value of this technology, we have created a panel of probes to target 401 human genes based on gene fusion events found in the Wellcome Trust databases [], Chimerdb 2.0 [] and The Cancer Genome Atlas () (full list in ) and have used this probe panel to identify known and previously unidentified gene fusion events in cancer cells lines and human whole brain RNA.
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Fig 1. Description of the Ovation Target Enrichment System.
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