An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples

<div><p>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 <i>in situ</i> 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.</p></div

EIF4E3-FOXP1 fusion transcript.

<p>(A) Structure of the fusion transcript based on UCSC genome browser tracks. Red arrow indicates the in frame fusion of EIF4E3 exon7 to FOXP1 exon 4. The Sanger sequencing trace below indicates the sequence of the fusion point, while the blue arrow over the sanger sequence trace indicates the 3’ end of the targeting probe. (B) RT-PCR result using PCR primers indicated described in S2 for detecting this fusion transcript. Lane 1: RT-PCR product showing the correct 209bp size, Lane 2: 50bp ladder.</p

HCC1937 Breast ductal carcinoma RNA.

<p>(A) FFPE RNA Bioanalyzer trace. (B) Bioanalyzer trace of sequencing library derived from 100 ng of RNA input shown in A. (C) Sequencing metrics for targeted RNA show that FFPE RNA is efficiently targeted.</p

Ovation Fusion Panel Target Enrichment System identifies known and novel gene fusion events in Universal Human Reference RNA.

<p>The number of sequencing reads determined to be derived from gene fusions in two different targeted sequencing libraries (blue and green) compared to the events identified in a standard, untargeted RNA-Seq library. The untargeted library (red) consists of 125 million total sequencing reads while the targeted libraries consist of 1.6 and 8.7 million sequencing reads.</p

Description of the Ovation Target Enrichment System.

<p>(A) Experimental steps of the assay and time required for each step. Adaptors (green) are ligated on to generated double stranded cDNA (ds-cDNA). Probes (shown in red and yellow) are hybridized to target cDNA and extended with a polymerase (dashed grey lines). All probes have common tail sequences (blue), which are used as priming sites along with adaptor sequences in subsequent library amplification PCR steps. (B) Example of probe positioning across different exons in a full length double stranded cDNA. Each exon (demarked by blue vertical lines) will have probes (green arrows; arrow points in the 3’ direction) designed to hybridize near the predicted exon-exon junctions. Exons larger than 300 nucleotides (nt) may have additional probes tiled along the length of the exon to obtain more complete sequence coverage. Probes are designed against both strands of the cDNA to enable identification of gene fusions when only one of the pair of genes is targeted. Translation start sequence (ATG) and poly A tail are labeled.</p