Single-cell individual full-length mtDNA sequencing by iMiGseq uncovers unexpected heteroplasmy shifts in mtDNA editing.

Nucleic acids research 2023 May 08

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The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed individual Mitochondrial Genome sequencing (iMiGseq) of full-length mtDNA for ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mtDNA molecule level. iMiGseq uncovered unappreciated levels of heteroplasmic variants in single cells well below the conventional NGS detection limit and provided accurate quantitation of heteroplasmy level. iMiGseq resolved the complete haplotype of individual mtDNA in single oocytes and revealed genetic linkage of de novo mutations. iMiGseq detected sequential acquisition of detrimental mutations, including large deletions, in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells. iMiGseq identified unintended heteroplasmy shifts in mitoTALEN editing, while showing no appreciable level of unintended mutations in DdCBE-mediated mtDNA base editing. Therefore, iMiGseq could not only help elucidate the mitochondrial etiology of diseases, but also evaluate the safety of various mtDNA editing strategies. © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

Citation

Chongwei Bi, Lin Wang, Yong Fan, Baolei Yuan, Gerardo Ramos-Mandujano, Yingzi Zhang, Samhan Alsolami, Xuan Zhou, Jincheng Wang, Yanjiao Shao, Pradeep Reddy, Pu-Yao Zhang, Yanyi Huang, Yang Yu, Juan Carlos Izpisua Belmonte, Mo Li. Single-cell individual full-length mtDNA sequencing by iMiGseq uncovers unexpected heteroplasmy shifts in mtDNA editing. Nucleic acids research. 2023 May 08;51(8):e48