An Affordable and Efficient In-House Approach for Detecting Gene Fusions in Bone and Soft Tissue Tumors Using a...

Accurate detection of gene fusions is critical for the diagnosis of bone and soft tissue tumors, yet conventional gene panel testing remains costly and impractical when processing small sample numbers. Here, we present a cost-effective and scalable in-house workflow using a custom capture panel combined with nanopore sequencing and TEQUILA-based probe synthesis. We analyzed 24 sarcoma samples with known gene fusions, comprising frozen and formalin-fixed paraffin-embedded (FFPE) specimens. Expected fusions were detected in all 7 frozen samples and in 16 of 17 FFPE samples. Long-read sequencing enabled direct characterization of full-length fusion transcripts, providing structural information beyond fusion junctions. Gene expression quantification showed strong concordance with short-read sequencing (Spearman r = 0.770-0.976), supporting quantitative reliability. Down-sampling analysis demonstrated that 100-200 Mb of sequencing data per sample was sufficient for reliable fusion detection, while deeper sequencing improved sensitivity for lowly expressed fusions. In addition, reuse of flow cells supported flexible and efficient sequencing for small sample batches. This approach provides a practical and extensible framework for in-house fusion detection and improves the accessibility of molecular diagnostics for rare cancers.