WITHDRAWN: NanoDel: a long-read sequencing pipeline for identifying large-scale mitochondrial DNA deletions validated in patient samples clinically diagnosed with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

Motivation Traditional methods for detecting large-scale mitochondrial DNA (mtDNA) deletions (LSMDs) in cells present challenges, i.e. a priori information, high DNA inputs, poor sensitivity and are not always quantitative. Mitigation can be achieved through high throughput DNA sequencing using e.g. Illumina and Oxford Nanopore Technologies (ONT), in combination with LSMD breakpoint identification and quantification using bioinformatic tools. Splice-aware RNA alignment tools increase the sensitivity for detecting LSMD breakpoints compared with DNA aligners. Long-read sequencing (LRS) also offers potential advantages over short read sequencing, e.g. greater read lengths and capturing variants on single reads. No existing pipelines capture the benefits of both a splice-aware alignment tool and LRS.

We developed “NanoDel”, a LRS pipeline, to sensitively and accurately detect cellular LSMDs. Using artificial datasets, “NanoDel” was more sensitive and accurate than other pipelines. In samples diagnosed with mitochondrial disease, it identified both known and previously uncharacterised (including mixtures) of LSMDs, without a priori information. LSMD breakpoints were found in mt-co1, mt-cyb, mt-nd6 and mt-nd5 genes. Analysis of selected LSMDs revealed proximity to repeat and putative G-quadruplex motifs, and occurrence in a range of healthy and pathological tissues, indicating potential for a shared vulnerability landscape in mtDNA, shaped by sequence motifs and structural constraints. “NanoDel” combined with one-amplicon, not two-amplicon, LR-PCR offers a robust strategy with clinical application for detecting LSMDs across a variety of cell/tissue samples, and it’s application across a broader range of samples, will yield new mechanistic insights into LSMD formation, and further our understanding of mtDNA instability. Competing Interest Statement The authors have declared no competing interest. Footnotes Availability and implementation: NanoDel is available at https://github.com/uopbioinformatics/NanoDel and raw read data are available through the NCBI Sequence Read Archive (SRA) under BioProject accession code PRJNA1369153 (https://www.ncbi.nlm.nih.gov/bioproject/1369153). Supplementary information: Supplementary data are available at Bioinformatics online. The revised version focusses solely on the development and validation of our novel bioinformatics approach. The molecular prognostic results: Figures 6-9, S5-S6, Tables S2C, and S6-S7 and associated text have been removed from version 1. This shift has enabled us to meet the strict word length constraints of the journal, while improving the flow of the manuscript and highlighting just the development and validation of our novel bioinformatics approach. To this end, Figure S1 from version 1 has been moved from SI to the main text, and Table S8 in version 1 has been updated. Also, a sizable proportion of materials and methods and results sections from the main text of version 1 have been updated and moved to SI. Taken together, these changes have necessitated figure and table numbering and author list modifications and have improved the clarity of our paper.