Single-cell full-length nanopore sequencing for quantitative variant analysis of native and genome-edited mitochondria
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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 detected sequential acquisition of detrimental mutations in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells (iPSCs). iMiGseq identified unintended heteroplasmy shifts in mitoTALEN edited NARP/Leigh syndrome iPSCs. iMiGseq of mitochondrial base editor DdCBE-edited cells did not detect any appreciable level of unintended mutations in mtDNA. iMiGseq uncovered unappreciated levels of heteroplasmic variants in single healthy human oocytes well below the conventional NGS detection limit, of which numerous variants are deleterious and associated with late-onset mitochondrial disease and cancer. iMiGseq revealed dramatic shifts in variant frequency and clonal expansion of large structural variants during oogenesis and stable heteroplasmy levels during human blastoid generation. It showed the first haplotype-resolved mitochondrial genomes from single human oocytes and single human blastoids.
Therefore, iMiGseq could not only help elucidate the mitochondrial etiology of diseases, but also enhance the precision of mitochondrial disease diagnosis.