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

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 scientists at NuGEN Technologies 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.

(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.

Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, the scientists 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.