Combining panel-based and whole-transcriptome-based gene fusion detection by long-read sequencing

Karleena Rybacki; Feng Xu; Hannah M Deutsch; Mian Umair Ahsan; Joe Chan; Zizhuo Liang; Yuanquan Song; Marilyn Li; Kai Wang

doi:10.1016/j.crmeth.2025.101111

Combining panel-based and whole-transcriptome-based gene fusion detection by long-read sequencing

Cell Rep Methods. 2025 Aug 18;5(8):101111. doi: 10.1016/j.crmeth.2025.101111. Epub 2025 Jul 21.

Authors

Karleena Rybacki¹, Feng Xu², Hannah M Deutsch³, Mian Umair Ahsan⁴, Joe Chan⁴, Zizhuo Liang⁴, Yuanquan Song⁵, Marilyn Li⁶, Kai Wang⁷

Affiliations

¹ Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
² Division of Genomic Diagnostics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
³ Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁴ Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
⁵ Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA; Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁶ Division of Genomic Diagnostics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁷ Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: wangk@chop.edu.

Abstract

We present a comprehensive gene fusion (GF) detection and analysis workflow that combines targeted panel-based and whole-transcriptome long-read sequencing. We first adapted libraries from the short-read CHOP Cancer Fusion Panel, which targets 119 oncogenes commonly implicated in cancer fusions, for use on Oxford Nanopore Technologies' long-read sequencing platform. Long-read sequencing successfully detected known GFs in panel-positive samples, confirming compatibility, and enabled reduced turnaround times. To expand GF discovery in clinically challenging cases, we analyzed 24 glioma samples with negative short-read fusion panel results using whole-transcriptome long-read sequencing. This identified 20 candidate GFs in panel-negative samples that were absent from current fusion databases, all of which were experimentally validated. In summary, we introduce a computational workflow that combines panel-based and whole-transcriptome long-read sequencing with tailored analysis pipelines to enable fast and comprehensive GF detection in cancer.

Keywords: CP: cancer biology; CP: genetics; Oxford Nanopore Technologies; computational pipeline; gene fusions; long-read sequencing; transcriptome analysis.

MeSH terms

Gene Expression Profiling* / methods
Gene Fusion* / genetics
Glioma / genetics
High-Throughput Nucleotide Sequencing* / methods
Humans
Transcriptome* / genetics