Abstract
The ontogeny and dynamics of mtDNA heteroplasmy remainunclear due to limitations of current mtDNA sequencing meth-ods. We developedindividualMitochondrialGenomesequen-cing (iMiGseq) of full-length mtDNA for ultra-sensitive variantdetection, complete haplotyping, and unbiased evaluation ofheteroplasmy levels, all at the individual mtDNA molecule level.iMiGseq detected sequential acquisition of detrimental muta-tions in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells (iPSCs). iMiGseq iden-tified unintended heteroplasmy shifts in mitoTALEN editedNARP/Leigh syndrome iPSCs. iMiGseq of mitochondrial baseeditor DdCBE-edited cells did not detect any appreciable levelof unintended mutations in mtDNA. iMiGseq uncovered unap-preciated levels of heteroplasmic variants in single healthy hu-man oocytes well below the conventional NGS detection limit,of which numerous variants are deleterious and associated withlate-onset mitochondrial disease and cancer. iMiGseq revealeddramatic shifts in variant frequency and clonal expansion oflarge structural variants during oogenesis and stable hetero-plasmy levels during human blastoid generation. It showed thefirst haplotype-resolved mitochondrial genomes from singlehuman oocytes and single human blastoids. Therefore, iMiGseqcould not only help elucidate the mitochondrial etiology ofdiseases, but also enhance the precision of mitochondrial diseasediagnosis.