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Bioinformatic Approaches to Study Mitochondrial DNA Heteroplasmy https://doi.org/10.17863/CAM.118291 Lyons, Camilla Chinnery, Patrick Mitochondria serve as signalling centres; they are primarily responsible for producing cellular energy and contain multiple copies of their own small, circular DNA, known as mitochondrial DNA(mtDNA). Mutations in mtDNA can be maternally inherited or somatic, when they occur after fertilisation. There are mechanisms at both the extra- and intracellular levels to prevent the transmission of these mutations across generations and cell cycles. The condition where both wild type and mutated mtDNA are present within the same cell is called hetero plasmy. Diverse and multi-system symptoms only appear when the level of mutated DNA becomes critical and exceeds above a threshold. While healthy individuals often carry low levels of mutations, primary mitochondrial diseases occur in patients with a high mutational load, affecting about 1 in 5,000 people. Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) is a primary mitochondrial disease that mainly affects the nervous system and muscles. It is most often caused by a heteroplasmic mutation at position m.3243A>G. To investigate the disease’s progression, we cultivated brain organoids from MELAS patients for either 100 or 200 days. Computational analysis of single cell RNA sequencing revealed the cellular mechanisms behind disease progression. We found evidence of calcium trafficking dysregu lation, which is associated with changes in mitochondrial morphology and function. This dysregulation can lead to axonal and dendritic abnormalities, predominantly affecting mature neurons and resulting in cellular developmental delays. Additionally, I address the main challenges of working with single-cell multiome data, with a focus on mitochondrial genomics. A key objective in the field is to estimate hetero plasmy at the single-cell level. The ATAC library of multiome experiments yields a large number of mitochondrial reads; however, determining the minimum mtDNA coverage needed to accurately estimate heteroplasmy remains to be defined. I approached this issue through comprehensive computational and statistical analysis, establishing standards that can benefit the entire research community. Finally, I developed a computational pipeline for analysing single-cell multiome datasets, with an emphasis on mitochondrial genomics. The advantages of a standardised pipeline include ensuring the reproducibility of results, as all software versions are documented. Additionally, the pipeline adheres to nf-core standards and is compartmentalised to prevent software incompatibilities. By parallelising processes and optimising computing resources, the overall run time of the pipeline is significantly reduced. I demonstrate the pipeline’s performance using both mouse and human test datasets. University of Cambridge 2024-09-27 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/384219 embargo 2026-05-19 https://www.repository.cam.ac.uk/bitstreams/d0145f6c-9de0-478a-91aa-58d9e862a652/download b1509544470e1c56e5acc3049b800381 https://www.repository.cam.ac.uk/bitstreams/16585325-7e0b-4610-b761-ed57c42fe347/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved mitochondria bioinformatics