NanoDel: a long-read sequencing pipeline for identifying large-scale mitochondrial DNA deletions validated in patient samples clinically diagnosed with mitochondrial disease and evaluated in glioblastoma

Primary glioblastoma is deadly, with limited improvements in prognosis. We developed “NanoDel”, a long-read sequencing pipeline to sensitively detect large-scale mitochondrial DNA (mtDNA) deletions (LSMDs) in glioblastoma to explore their prognostic significance. Following validation using artificial datasets and samples from patients diagnosed with mitochondrial disease, “NanoDel” identified 171 low-level heterogeneous glioblastoma-LSMDs, clustering in mt-co1, mt-cyb genes, and adjacent to repeat, and G4 motifs. These “hotspots” were also seen in other tissues, revealing a shared vulnerability in mtDNA. While LSMD burden alone was not prognostic, mtDNA copy number and complex IV activity strongly stratified glioblastoma patient survival. “NanoDel” offers a clinically and cross-organismal applicable tool for LSMD detection and new insights into mtDNA markers and instability mechanisms.