Characterization of Large-Scale Mitochondrial DNA Rearrangements Using Complementary Short- and Long-Read Sequencing Approaches.
6 affiliations
- 1Division of Genomic Diagnostics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
- 2Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
- 3Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
- 4Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
- 5Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
- 6Center for Mitochondrial and Epigenomic Medicine, Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Abstract
Background
Large-scale mitochondrial DNA (mtDNA) rearrangement, including deletion or duplication, is one of the important causes of primary mitochondrial disease. In clinical practice, short-read sequencing (SRS)-based assays typically detect these rearrangements by identifying regional mtDNA copy-number changes but generally do not directly distinguish deletions from duplications. As a result, mtDNA duplications may be misclassified as deletions, potentially leading to inaccurate molecular diagnoses and genotype-phenotype correlations. This study aimed to evaluate the utility of long-read sequencing (LRS) for the definitive characterization of large-scale mtDNA rearrangements.Methods
Two individuals who had been reported to possess single large-scale mtDNA deletions using SRS-based clinical tests were re-evaluated because their clinical presentations were not consistent with typical mtDNA deletion syndromes. Existing SRS data were reanalyzed, and the suspected rearrangements were independently characterized using both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies long-read sequencing platforms.Results
Reanalysis of SRS data suggested that the reported deletions were more consistent with mtDNA duplications. This finding was confirmed by both PacBio and Oxford Nanopore LRS in one case. Reclassifying the deletion to a duplication in this case substantially improved genotype-phenotype interpretation and better explained the atypical clinical features observed in this individual. However, LRS could not capture sufficient long reads that give direct evidence of duplication in the second case due to low heteroplasmy of the mtDNA structural variant. Therefore, this case remains inconclusive.Conclusions
SRS and LRS provide complementary information for the evaluation of large-scale mtDNA rearrangements. SRS can sensitively identify mtDNA large deletions, but it cannot distinguish mtDNA deletion from duplication. On the other hand, LRS can provide direct structural information and confirm duplication. However, LRS may have limited sensitivity for low-level heteroplasmic structural variants. An integrated approach incorporating both methods, together with clinical correlation and reanalysis of potentially discordant findings, may improve the characterization and interpretation of mtDNA rearrangements.
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BibTeX
@article{wang2026characterization,
title = {{Characterization of Large-Scale Mitochondrial DNA Rearrangements Using Complementary Short- and Long-Read Sequencing Approaches.}},
author = {Jing Wang and Joe Chan and Mian Umair Ahsan and Amy Goldstein and Rebecca D Ganetzky and Sonal Sharma and James T Peterson and Douglas C Wallace and Tanaya Jadhav and Ramakrishnan Rajagopalan and Kai Wang},
journal = {Genes},
year = {2026},
month = sep,
doi = {10.3390/genes17101161},
url = {https://europepmc.org/article/MED/42938277},
note = {CC BY},
}
RIS
TY - JOUR
TI - Characterization of Large-Scale Mitochondrial DNA Rearrangements Using Complementary Short- and Long-Read Sequencing Approaches.
AU - Jing Wang
AU - Joe Chan
AU - Mian Umair Ahsan
AU - Amy Goldstein
AU - Rebecca D Ganetzky
AU - Sonal Sharma
AU - James T Peterson
AU - Douglas C Wallace
AU - Tanaya Jadhav
AU - Ramakrishnan Rajagopalan
AU - Kai Wang
PY - 2026
DA - 2026/09/22
JO - Genes
DO - 10.3390/genes17101161
UR - https://europepmc.org/article/MED/42938277
AB - Background Large-scale mitochondrial DNA (mtDNA) rearrangement, including deletion or duplication, is one of the important causes of primary mitochondrial disease. In clinical practice, short-read sequencing (SRS)-based assays typically detect these rearrangements by identifying regional mtDNA copy-number changes but generally do not directly distinguish deletions from duplications. As a result, mtDNA duplications may be misclassified as deletions, potentially leading to inaccurate molecular diagnoses and genotype-phenotype correlations. This study aimed to evaluate the utility of long-read sequencing (LRS) for the definitive characterization of large-scale mtDNA rearrangements.Methods Two individuals who had been reported to possess single large-scale mtDNA deletions using SRS-based clinical tests were re-evaluated because their clinical presentations were not consistent with typical mtDNA deletion syndromes. Existing SRS data were reanalyzed, and the suspected rearrangements were independently characterized using both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies long-read sequencing platforms.Results Reanalysis of SRS data suggested that the reported deletions were more consistent with mtDNA duplications. This finding was confirmed by both PacBio and Oxford Nanopore LRS in one case. Reclassifying the deletion to a duplication in this case substantially improved genotype-phenotype interpretation and better explained the atypical clinical features observed in this individual. However, LRS could not capture sufficient long reads that give direct evidence of duplication in the second case due to low heteroplasmy of the mtDNA structural variant. Therefore, this case remains inconclusive.Conclusions SRS and LRS provide complementary information for the evaluation of large-scale mtDNA rearrangements. SRS can sensitively identify mtDNA large deletions, but it cannot distinguish mtDNA deletion from duplication. On the other hand, LRS can provide direct structural information and confirm duplication. However, LRS may have limited sensitivity for low-level heteroplasmic structural variants. An integrated approach incorporating both methods, together with clinical correlation and reanalysis of potentially discordant findings, may improve the characterization and interpretation of mtDNA rearrangements.
SN - 2073-4425
ER -