Fusion transcripts and their genomic breakpoints in polyadenylated and ribosomal RNA–minus RNA sequencing data

Abstract

Background Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non–poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints. Results We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA–minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG–positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq. Conclusion By using the full potential of non–poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects.

CRISPRs in the human genome are differentially expressed between malignant and normal adjacent to tumor tissue

“…In the publicly available whole-transcriptome sequencing dataset (NGS-ProToCol EGAS00001002816) 40 – 42 , which includes 50 malignant and 40 normal adjacent to tumor prostate tissues, 177 hCRISPRs revealed a substantial difference in expression levels between malignant and normal adjacent to tumor prostate tissue (Fig. 5a, b, c and Supplementary Data 1 ).…”

“…This opens the door to explore the usage of hCRISPRs in diagnostics and prognostics to detect a wide variety of diseases, including cancer. In prostate cancer, we already made advantage of this discovery, as we identified 115 hCRISPRs (nine without any overlapping gene) that are candidate biomarkers as validated in two independent prostate cancer related datasets (Mitranscriptome and NGS-ProToCol) 40 , 42 – 44 , 62 . For two of these hCRISPRs (chr9_209 and chr19_106), we further performed and established the ability to distinguish malignant from normal adjacent to tumor prostate tissue by RT-qPCR, confirming whole-transcriptome results of the same tissues reported earlier 38 , 39 .…”

Novel gene–intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy

“…BCR-ABL 88 , 89 and TMPRSS-ERG 90 ) resulting in chimeric mRNA transcripts that can form oncogenic ‘neo-antigens’. 96 Fusions involving intergenic/non-coding sequences that result in cryptic (pseudo) exon formation are by comparison rare, only being reported by a few studies. 96–99 Interestingly, the majority of genomic breakpoints in fusion genes are intergenic or intronic and are therefore not typically present in mRNA or protein-coding sequences, 93 , 94 , 100 making detection of gene–intergenic fusions difficult.…”

Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy: A new mechanism for motor neuron degeneration

“…BCR-ABL 85,86 and TMPRSS-ERG 87 ) resulting in chimeric mRNA transcripts that can form oncogenic “neo-antigens”. 93 Fusions involving intergenic/non-coding sequences that result in cryptic (pseudo) exon formation are by comparison rare – only being reported by a few studies. 93–96 Interestingly, the majority of genomic breakpoints in fusion genes are intergenic or intronic and are therefore not typically present in mRNA or protein-coding sequences 90,91,97 making detection of gene-intergenic fusions difficult.…”

“…93 Fusions involving intergenic/non-coding sequences that result in cryptic (pseudo) exon formation are by comparison rare – only being reported by a few studies. 93–96 Interestingly, the majority of genomic breakpoints in fusion genes are intergenic or intronic and are therefore not typically present in mRNA or protein-coding sequences 90,91,97 making detection of gene-intergenic fusions difficult. This may account for the predominance of classical gene-gene fusions over gene-intergenic fusions in the literature to date.…”