Chimeric transcript discovery by paired-end transcriptome sequencing

Abstract

Recurrent gene fusions are a prevalent class of mutations arising from the juxtaposition of 2 distinct regions, which can generate novel functional transcripts that could serve as valuable therapeutic targets in cancer. Therefore, we aim to establish a sensitive, high-throughput methodology to comprehensively catalog functional gene fusions in cancer by evaluating a paired-end transcriptome sequencing strategy. Not only did a paired-end approach provide a greater dynamic range in comparison with single read based approaches, but it clearly distinguished the high-level "driving" gene fusions, such as BCR-ABL1 and TMPRSS2-ERG, from potential lower level "passenger" gene fusions. Also, the comprehensiveness of a paired-end approach enabled the discovery of 12 previously undescribed gene fusions in 4 commonly used cell lines that eluded previous approaches. Using the paired-end transcriptome sequencing approach, we observed read-through mRNA chimeras, tissue-type restricted chimeras, converging transcripts, diverging transcripts, and overlapping mRNA transcripts. Last, we successfully used paired-end transcriptome sequencing to detect previously undescribed ETS gene fusions in prostate tumors. Together, this study establishes a highly specific and sensitive approach for accurately and comprehensively cataloguing chimeras within a sample using paired-end transcriptome sequencing.

Fig. 1.

Dynamic range and sensitivity of the paired-end transcriptome analysis relative to single read approaches. (A) Comparison of paired-end (blue) and long single transcriptome reads (black) supporting known gene fusions TMPRSS2-ERG, BCR-ABL1, BCAS4-BCAS3, and ARFGEF2-SULF2. (B) Schematic representation of TMPRSS2-ERG in VCaP, comparing mate pairs with long single transcriptome reads. (Upper) Frequency of mate pairs, shown in log scale, are divided based on whether they encompass or span the fusion boundary; (Lower) 100-mer single transcriptome reads spanning TMPRSS2-ERG fusion boundary. First 36 nt are highlighted in red. (C) Venn diagram of chimera nominations from both a paired-end (orange) and long single read (blue) strategy for UHR and HBR.

Fig. 2.

RNA based chimeras. (A) Heatmaps showing the normalized number of reads supporting each read-through chimera across samples ranging from 0 (white) to 30 (red). (Upper) The heatmap highlights broadly expressed chimeras in UHR, HBR, VCaP, and K562. (Lower) The heatmap highlights the expression of the top ranking restricted gene fusions that are enriched with interchromosomal and intrachromosomal rearrangements. (B) Illustrative examples classifying RNA-based chimeras into (i) read-throughs, (ii) converging transcripts, (iii) diverging transcripts, and (iv) overlapping transcripts. (C Upper) Paired-end approach links reads from independent genes as belonging to the same transcriptional unit (Right), whereas a single read approach would assign these reads to independent genes (Left). (Lower) The single read approach requires that a chimera span the fusion junction (Left), whereas a paired-end approach can link mate pairs independent of gene annotation (Right).

Fig. 3.

Discovery of previously undescribed ETS gene fusions in localized prostate cancer. (A) Schematic representation of the interchromosomal gene fusion between exon 1 of HERPUD1 (red), residing on chromosome 16, with exon 4 of ERG (blue), located on chromosome 21. (B) Schematic representation showing genomic organization of HERPUD1 and ERG genes. Horizontal red and green bars indicate the location of BAC clones. (Lower) FISH analysis using BAC clones showing HERPUD1 and ERG in a normal tissue (Left), deletion of the ERG 5′ region in tumor (Center), and HERPUD1-ERG fusion in a tumor sample (Right). (C) Schematic representation of the interchromosomal gene fusion between FLJ35294 (green), residing on chromosome 17, with exon 4 of ETV1 (orange) located on chromosome 21. (D Upper) Schematic representation of the genomic organization of FLJ35294 and ETV1 genes. (Lower) FISH analysis using BAC clones showing split of ETV1 in tumor sample (Left) and the colocalization of FLJ35294 and ETV1 in a tumor sample (Right).