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Updated: Dec 16, 2025
Rup (RNA-seq Usability Assessment Pipeline) - Quality Control for Bulk RNA-seq Experiments in Eukaryotes
Published on: November 7, 2025
Key considerations for comprehensive validation of an RNA fusion NGS panel
Subit Barua1, Gary Wang1, Mahesh Mansukhani1
1Department of Pathology and Cell Biology, Columbia University Medical Center, New York, USA.
Objectives:
Validation of RNA-based NGS assays for the detection of therapeutically targetable gene fusions is challenging. Here, we report systematic validation and quality control monitoring of our targeted fusion panel for the detection of 17 clinically relevant fusion transcripts across several tumor types. We implemented this RNA Fusion Panel as a reflex test for tumors lacking DNA driver mutations.
Design:
Forty-four formalin-fixed, paraffin-embedded (FFPE) or fresh-frozen lung, brain, soft tissue and skin tumors were used to determine the accuracy of the assay. Additional fusion-positive specimens and a calibrated reference standard were used to establish the precision, reproducibility and sensitivity of the assay. All aspects of the validation, including quality control metrics, were performed according to New York State guidelines.
Results:
For the RNA fusion panel, accuracy, reproducibility and precision studies were above 99%. Reproducibility and sensitivity studies with the reference standard were helpful in identifying inconsistencies. The limit of detection for most RNA fusion transcripts was 50 copies. Application of the RNA fusion assay as a reflex test to 450 tumor samples lacking DNA driver mutations resulted in a 10% increase in diagnostic yield with minimal additional processing time.
Conclusions:
The validated RNA fusion panel provides clinical utility in therapy selection for patients with solid tumors. By using a sequential testing approach, the RNA fusion assay complements the DNA hotspot assay in identifying clinically relevant variants across many tumor types with minimal additional increase in processing time.
Insights
This study validates an RNA fusion panel for detecting gene fusions in solid tumors, improving diagnostic yield by 10% when used as a reflex test. The assay demonstrates high accuracy and clinical utility for therapy selection.
Area of Science:
- Molecular diagnostics
- Genomic medicine
- Oncology
Background:
- Detecting therapeutically targetable gene fusions using RNA-based next-generation sequencing (NGS) assays presents validation challenges.
- Targeted fusion panels are crucial for identifying actionable mutations in solid tumors.
Purpose of the Study:
- To systematically validate a targeted RNA fusion panel for detecting 17 clinically relevant gene fusions.
- To implement and assess the utility of this RNA Fusion Panel as a reflex test for tumors lacking DNA driver mutations.
Main Methods:
- Validation involved 44 formalin-fixed, paraffin-embedded (FFPE) or fresh-frozen tumor samples across various types.
- Accuracy, precision, reproducibility, and sensitivity were assessed using additional fusion-positive specimens and a calibrated reference standard.
- Quality control metrics adhered to New York State guidelines.
Main Results:
- The RNA fusion panel achieved accuracy, reproducibility, and precision rates exceeding 99%.
- The limit of detection for most RNA fusion transcripts was 50 copies.
- Implementing the assay as a reflex test increased diagnostic yield by 10% in 450 tumor samples with minimal added processing time.
Conclusions:
- The validated RNA fusion panel offers significant clinical utility for guiding therapy selection in patients with solid tumors.
- This RNA fusion assay complements DNA hotspot testing in a sequential approach, enhancing the identification of clinically relevant variants.
- The assay provides a valuable tool for comprehensive genomic profiling across diverse tumor types.
