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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Updated: Nov 18, 2025

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
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Gene Fusion Identification Using Anchor-Based Multiplex PCR and Next-Generation Sequencing.

Yu-Wei Cheng1, Anders Meyer2,3, Maureen A Jakubowski1

  • 1Department of Laboratory Medicine, Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH.

The Journal of Applied Laboratory Medicine
|February 4, 2021
PubMed
Summary

A new next-generation sequencing (NGS) panel accurately detects gene fusions in soft-tissue sarcomas. This validated test offers high sensitivity and specificity for clinical use.

Keywords:
bone and soft-tissue neoplasmsformalin-fixed paraffin-embedded (FFPE) tissuegene fusionneurotrophin receptor tyrosine kinase (NTRK)next-generation sequencing

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Area of Science:

  • Oncology
  • Genetics
  • Molecular Diagnostics

Background:

  • Current gene fusion detection methods (FISH, IHC, transcriptome analysis) have limitations.
  • Bioinformatics expertise is often required, hindering clinical laboratory application.
  • Soft-tissue sarcomas frequently involve gene fusions, necessitating reliable detection methods.

Purpose of the Study:

  • To analytically validate a customized next-generation sequencing (NGS) panel for detecting gene fusions in 34 genes relevant to soft-tissue sarcomas.
  • To assess the feasibility of NGS-based fusion detection in routine clinical laboratories.

Main Methods:

  • Validated a customized NGS panel targeting 34 genes for fusion events.
  • Utilized 87 formalin-fixed paraffin-embedded (FFPE) tissues with known gene fusion status.
  • Employed RNA-level analysis with gene-specific primers, PCR enrichment, and bioinformatics analysis.

Main Results:

  • Established quality metrics for fusion detection, including nucleic acid quantity and sequencing fragment counts.
  • Achieved analytical sensitivity of 98.7% and analytical specificity of 90.0%.
  • Demonstrated high concordance between NGS panel results and alternative testing methods.

Conclusions:

  • The validated NGS panel provides a sensitive and specific method for detecting gene fusions in soft-tissue sarcomas.
  • This NGS approach is suitable for clinical laboratories, overcoming limitations of existing methods.
  • The panel offers a reliable tool for molecular diagnosis and targeted therapy selection in sarcoma patients.