Dataset Information

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

ABSTRACT: 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. 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. 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.

SUBMITTER: Hoogstrate Y

PROVIDER: S-EPMC8673554 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Hoogstrate Youri Y Komor Malgorzata A MA Böttcher René R van Riet Job J van de Werken Harmen J G HJG van Lieshout Stef S Hoffmann Ralf R van den Broek Evert E Bolijn Anne S AS Dits Natasja N Sie Daoud D van der Meer David D Pepers Floor F Bangma Chris H CH van Leenders Geert J L H GJLH Smid Marcel M French Pim J PJ Martens John W M JWM van Workum Wilbert W van der Spek Peter J PJ Janssen Bart B Caldenhoven Eric E Rausch Christian C de Jong Mark M Stubbs Andrew P AP Meijer Gerrit A GA Fijneman Remond J A RJA Jenster Guido W GW

GigaScience 20211201 12

<h4>Background</h4>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.<h4>Results</h4>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 no ...[more]

PMID: 34891161

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Project description:Herpesviruses employ extensive bidirectional transcription of overlapping genes to overcome length constraints on their gene product repertoire. As a consequence, many lytic transcripts cannot be measured individually by reverse transcription-quantitative PCR (RT-qPCR) or conventional RNA sequencing (RNA-seq) analysis. A. G. Bruce, S. Barcy, T. DiMaio, E. Gan, et al. (Pathogens 6:11, 2017, https://doi.org/10.3390/pathogens6010011) have proposed an approximation method using unique coding sequences (UCDS) to estimate lytic gene abundance from Kaposi's sarcoma-associated herpesvirus (KSHV) RNA-seq data. Although UCDS has been widely employed, its accuracy, to our knowledge, has never been rigorously validated for any herpesvirus. In this study, we use cap analysis of gene expression sequencing (CAGE-seq) as a gold-standard to determine the accuracy of UCDS for estimating Epstein-Barr virus (EBV) lytic gene expression levels from RNA-seq data. We also introduce the Unique TranScript (UTS) method, which, like UCDS, estimates transcript abundance from changes in mean RNA-seq read depth. UTS is distinguished by its use of empirically determined 5' and 3' transcript ends rather than coding sequence annotations. Compared to conventional read assignment, both UCDS and UTS improved the accuracy of quantitation of overlapping genes, with UTS giving the most-accurate results. The UTS method discards fewer reads and may be advantageous for experiments with less sequencing depth. UTS is compatible with any aligner and, unlike isoform-aware alignment methods, can be implemented on a laptop computer. Our findings demonstrate that the accuracy achieved by complex and expensive techniques such as CAGE-seq can be approximated using conventional short-read RNA-seq data when read assignment methods address transcript overlap. Although our study focuses on EBV transcription, the UTS method should be applicable across all herpesviruses as well as to other genomes with extensively overlapping transcriptomes. IMPORTANCE Many viruses employ extensively overlapping transcript structures. This complexity makes it difficult to quantify gene expression by using conventional methods, including RNA-seq. Although high-throughput techniques that overcome these limitations exist, they are complex, expensive, and scarce in the herpesvirus literature relative to short-read RNA-seq. Here, using Epstein-Barr virus (EBV) as a model, we demonstrate that conventional RNA-seq analysis methods fail to accurately quantify the abundances of many overlapping transcripts. We further show that the previously described Unique CoDing Sequence (UCDS) method and our Unique TranScript (UTS) method greatly improve the accuracy of EBV lytic gene measurements obtained from RNA-seq data. The UTS method has the advantages of discarding fewer reads and being implementable on a laptop computer. Although this study focuses on EBV, the UCDS and UTS methods should be applicable across herpesviruses and for other viruses that make extensive use of overlapping transcription.

Dataset Information

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

Publications

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

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