e20042 Background: In non-small cell lung cancer (NSCLC), more and more driver fusions are suitable for targeted therapy. Sequential or parallel DNA and RNA sequencing is an option, but it is costly and requires sufficiently high quality biopsy material. The purpose of this study was to confirm the consistency of fusion genes detected by DNA-based NGS and RNA-based NGS in the same sample, and to validate the performance of FFPE samples for RNA-based NGS. Methods: In this study, we evaluated the benefits of capture-based DNA sequencing and amplicon-based RNA sequencing in identifying gene fusion and exon-skipping events in 98 NSCLC patients. All cases were FFPE samples stored for 1-5 years. In addition, we investigated the limiting performance of RNA-based NGS for RNA initiation. Results: In the cases of positive DNA sequencing fusion samples, the fusion consistency detection rate of RNA sequencing was 95% (38/40). Two cases were not detected by RNA-based NGS, one was a five-year FFPE sample and the other was due to a probe not covered problem. In patients with negative DNA fusion genes, RNA sequencing could detect 12.07% more fusion-positive cases involving ALK (n = 1), ROS (n = 3), MET 14 exon skipping (n = 1), RET (n = 1), and BRAF (n = 1). Moreover, the FFPE samples stored for 3-5 years inevitably degrade the RNA, but could still be used for RNA-based sequencing. Among 66 FFPE samples stored for 3-5 years, the detection rate of fusion consistency between DNA sequencing fusion samples and RNA sequencing was 96.77%(30/31). In patients with negative DNA fusion genes, RNA sequencing was able to detect 14.29% (5/35) more fusion-positive cases. The performance verification of the other 8 samples with different RNA input amounts found that 87.5% (7/8) of the samples could meet the quality control of RNA sequencing and accurately detect fusion mutations as low as 1 ng. Conclusions: Molecular profiling based on mixed DNA and RNA sequencing improves the effectiveness of detection of fusion-driven NSCLC. The method was feasible on small tissue samples as well as FFPE samples stored for 3-5 years, and could greatly reduce the complexity and cost of molecular examination.