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Updated: Oct 12, 2025
Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Published on: August 4, 2016
Transcript-targeted analysis reveals isoform alterations and double-hop fusions in breast cancer
Shinichi Namba1,2, Toshihide Ueno1, Shinya Kojima1
1Division of Cellular Signaling, National Cancer Center Research Institute, Tokyo, 104-0045, Japan.
Abstract:
Although transcriptome alteration is an essential driver of carcinogenesis, the effects of chromosomal structural alterations on the cancer transcriptome are not yet fully understood. Short-read transcript sequencing has prevented researchers from directly exploring full-length transcripts, forcing them to focus on individual splice sites. Here, we develop a pipeline for Multi-Sample long-read Transcriptome Assembly (MuSTA), which enables construction of a transcriptome from long-read sequence data. Using the constructed transcriptome as a reference, we analyze RNA extracted from 22 clinical breast cancer specimens. We identify a comprehensive set of subtype-specific and differentially used isoforms, which extended our knowledge of isoform regulation to unannotated isoforms including a short form TNS3. We also find that the exon-intron structure of fusion transcripts depends on their genomic context, and we identify double-hop fusion transcripts that are transcribed from complex structural rearrangements. For example, a double-hop fusion results in aberrant expression of an endogenous retroviral gene, ERVFRD-1, which is normally expressed exclusively in placenta and is thought to protect fetus from maternal rejection; expression is elevated in several TCGA samples with ERVFRD-1 fusions. Our analyses provide direct evidence that full-length transcript sequencing of clinical samples can add to our understanding of cancer biology and genomics in general.
Insights
This study introduces Multi-Sample long-read Transcriptome Assembly (MuSTA) to analyze full-length cancer transcripts. MuSTA reveals novel cancer-specific isoforms and fusion transcripts, advancing our understanding of cancer genomics.
Area of Science:
- Genomics
- Cancer Biology
- Transcriptomics
Background:
- Transcriptome alterations are key drivers of cancer, but chromosomal structural changes' impact remains unclear.
- Short-read sequencing limits analysis to splice sites, hindering full-length transcript exploration.
Purpose of the Study:
- To develop a pipeline for long-read transcriptome assembly from multiple samples.
- To analyze clinical breast cancer specimens using this pipeline to identify novel transcript isoforms and fusion events.
Main Methods:
- Development of the Multi-Sample long-read Transcriptome Assembly (MuSTA) pipeline.
- Analysis of RNA from 22 clinical breast cancer specimens using long-read sequencing.
- Identification and characterization of subtype-specific and differentially used isoforms, including unannotated ones.
Main Results:
- Identification of a comprehensive set of subtype-specific and differentially used isoforms, extending knowledge to unannotated isoforms like TNS3.
- Discovery that fusion transcript exon-intron structure is context-dependent.
- Identification of double-hop fusion transcripts arising from complex rearrangements, including one leading to aberrant ERVFRD-1 expression.
Conclusions:
- Full-length transcript sequencing of clinical samples provides crucial insights into cancer biology and genomics.
- MuSTA enables a deeper understanding of isoform regulation and complex genomic alterations in cancer.
- Findings highlight the significance of structural variations in shaping the cancer transcriptome.
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