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Pilot-study for a novel universal tool for detection of oncogenic fusion transcripts

ABSTRACT: A novel oligonucleotide microarray design is described whereby one can screen for all known oncogenic fusion transcripts by one microarray hybridization. Measurements of chimeric transcript junctions are combined with exon-wise measurements of individual fusion partners. Keywords: Nimblegen custom-design The pilot data included a design with 68,861 oligonucleotide probes covering all combinations of chimeric exon-exon junctions from 275 pairs of fusion genes, as well as sets of oligos internal to all the exons of the fusion partners. Proof of principle was demonstrated by detection of known fusion genes (such as TCF3:PBX1, ETV6:RUNX1, and TMPRSS2:ERG) from six positive controls consisting of leukemia cell lines and prostate cancer biopsies.

ORGANISM(S): Homo sapiens

SUBMITTER: Guro Lind

PROVIDER: E-GEOD-14435 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A universal assay for detection of oncogenic fusion transcripts by oligo microarray analysis.

Skotheim Rolf I RI Thomassen Gard O S GO Eken Marthe M Lind Guro E GE Micci Francesca F Ribeiro Franclim R FR Cerveira Nuno N Teixeira Manuel R MR Heim Sverre S Rognes Torbjørn T Lothe Ragnhild A RA

Molecular cancer 20090119

<h4>Background</h4>The ability to detect neoplasia-specific fusion genes is important not only in cancer research, but also increasingly in clinical settings to ensure that correct diagnosis is made and the optimal treatment is chosen. However, the available methodologies to detect such fusions all have their distinct short-comings.<h4>Results</h4>We describe a novel oligonucleotide microarray strategy whereby one can screen for all known oncogenic fusion transcripts in a single experiment. To a ...[more]

PMID: 19152679

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Pilot-study for a novel universal tool for detection of oncogenic fusion transcripts

Project description:A novel oligonucleotide microarray design is described whereby one can screen for all known oncogenic fusion transcripts by one microarray hybridization. Measurements of chimeric transcript junctions are combined with exon-wise measurements of individual fusion partners. Keywords: Nimblegen custom-design

2009-01-19 | GSE14435 | GEO

Antibody detection of translocations (ADOT) in Ewing Sarcoma

Project description:We developed a new approach called antibody detection of translocations (ADOT) which combines a transcriptional microarray-based approach with a novel antibody-based detection method to detect translocations in cancer. ADOT allows for the accurate and sensitive identification of translocations and provides exon-level information about the fusion transcript. ADOT can detect translocations in poor quality unprocessed total RNA. We demonstrate the feasibility of ADOT by examples in which both known and unknown Ewing sarcoma translocations are identified from cell lines, tumor xenografts, and FFPE primary tumors. These results demonstrate that ADOT may be an effective approach for translocation analysis in clinical specimens with significant RNA degradation and may offer a novel diagnostic tool for translocation-based cancers. We designed oligonucleotide probes for each possible exon-exon combination between potential fusion partners and printed the DNA oligonucleotides on custom-designed microarrays. Total RNA from tumor cells or tissues was hybridized on the array. Bound RNA was detected with the S9.6 monoclonal antibody that recognizes RNA-DNA duplexes in a sequence-independent fashion,and detected with Cy3-labeled anti-mouse IgG.

2012-01-31 | E-GEOD-35450 | biostudies-arrayexpress

Large scale screening for known fusion genes in cancer cell lines

Project description:We have utilized a version 2.0 of our Fusion gene microarray (version 1.0 described by PMID: 19152679 and GEO, GPL8078) to screen cancer cell lines for known fusion genes We assembled a comprehensive database of published fusion genes, including those which have been reported only in individual studies and samples, and fusion genes resulting from deep sequencing of cancer genomes and transcriptomes. From the total set of 548 fusion genes, we designed 197,846 unique oligonucleotides, targeting both chimeric transcript junctions as well as sequences internal to each of the fusion gene partners. We investigated the presence of fusion genes in a series of 67 cell lines originating from 15 different cancer types. Data from ten leukaemia cell lines with known fusion gene status were used to evaluate and calibrate an automated scoring algorithm. In five out of ten cell lines the correct fusion gene was the top most scoring hit, and one came second, all six passing a cut-off of 50 percent of theoretical maximum-score. Two additional fusion genes, BCAS4-BCAS3 from the MCF-7 breast cancer cell line and CCDC6-RET from the TPC-1 thyroid cancer cell line were validated as true positive fusion transcripts from the remaining 57 cell lines. A total of 67 cell lines from various cancer types were analysed for presence of known fusion genes

2011-02-10 | E-GEOD-24737 | biostudies-arrayexpress

Project description:Sequencing of chimeric deletion junctions

| PRJNA1117804 | ENA

The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

Project description:To uncover exon junction complex (EJC) deposition sites on cellular mRNAs, RNA footprints of EJC immuo-purified from HEK293 cells were deep sequenced. The analysis of these data revealed that major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions (-24 position) and that the majority of exon junctions carry an EJC. Unexpectedly, we find that many sites further upstream of -24 position are also enriched in these EJC footprints. These "non-canonical" sites are binding sites of EJC-interacting proteins with a subset being occupied by SR proteins. Thus, an EJC-SR protein nexus exists within spliced mRNPs and is revealed here. Deep sequencing based profiling of EJC RNA footprints obtained by tandem RNA immunoprecipitation (RIPiT) of RNase I digested RNA:protein complexes.

2012-10-18 | E-GEOD-41154 | biostudies-arrayexpress

Large scale screening for known fusion genes in cancer cell lines

2011-02-10 | GSE24737 | GEO

Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox [Screen]

Project description:Alternative splicing is an essential mechanism for diversifying proteins, with different mature RNA isoforms producing different proteins with potentially distinct functions. Two major challenges in characterizing the cellular function of isoforms are the lack of both experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design. To address these gaps, we developed and methodically tested a strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs (gRNAs) that overlap exon-exon junctions in the mature RNA. We perform a high-throughput essentiality screen, numerous qPCR assays, and PacBio long read sequencing to affirm that our strategy works to specifically target the unique junctions of isoforms to robustly knockdown their RNA. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions in current GENCODE annotations and our predictions of gRNA efficacy, we estimate that this strategy can target 94% of all human isoforms, including 29,238 protein-coding and 9,638 lncRNA isoforms that are specifically targetable with a gRNA spanning a unique splice junction.

2023-09-13 | GSE242106 | GEO

Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox [LR-RNA-Seq]

2023-09-13 | GSE242104 | GEO

Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox [RNA-seq]

2023-09-13 | GSE242105 | GEO

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation

Project description:Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

2024-06-18 | PXD052856 | Pride

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