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Updated: Aug 4, 2025
Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
Published on: July 12, 2022
Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in Human single oocytes, blastoids, and
Chongwei Bi1, Lin Wang1, Yong Fan2
1Bioscience program, Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia.
Abstract:
Maternal mitochondria are the sole source of mtDNA for every cell of the offspring. Heteroplasmic mtDNA mutations inherited from the oocyte are a common cause of metabolic diseases and associated with late-onset diseases. However, the origin and dynamics of mtDNA heteroplasmy remain unclear. We used our individual Mitochondrial Genome sequencing (iMiGseq) technology to study mtDNA heterogeneity, quantitate single nucleotide variants (SNVs) and large structural variants (SVs), track heteroplasmy dynamics, and analyze genetic linkage between variants at the individual mtDNA molecule level in single oocytes and human blastoids. Our study presented the first single-mtDNA analysis of the comprehensive heteroplasmy landscape in single human oocytes. Unappreciated levels of rare heteroplasmic variants well below the detection limit of conventional methods were identified in healthy human oocytes, of which many are reported to be deleterious and associated with mitochondrial disease and cancer. Quantitative genetic linkage analysis revealed dramatic shifts of variant frequency and clonal expansions of large SVs during oogenesis in single-donor oocytes. iMiGseq of a single human blastoid suggested stable heteroplasmy levels during early lineage differentiation of naïve pluripotent stem cells. Therefore, our data provided new insights of mtDNA genetics and laid a foundation for understanding mtDNA heteroplasmy at early stages of life.
Insights
Maternal mitochondrial DNA (mtDNA) heteroplasmy in oocytes harbors rare, deleterious variants. Our novel sequencing technology reveals dynamic shifts during oogenesis, impacting early life development and disease risk.
Area of Science:
- Genetics and Genomics
- Mitochondrial Biology
- Reproductive Biology
Background:
- Maternal mitochondria are the sole source of mitochondrial DNA (mtDNA) for offspring.
- Inherited mtDNA heteroplasmy from oocytes is linked to metabolic and late-onset diseases.
- The origins and dynamics of mtDNA heteroplasmy are not fully understood.
Purpose of the Study:
- To investigate mtDNA heterogeneity and heteroplasmy dynamics at the single-molecule level in human oocytes and blastoids.
- To quantify single nucleotide variants (SNVs) and large structural variants (SVs) in mtDNA.
- To analyze genetic linkage between variants and track heteroplasmy changes during early development.
Main Methods:
- Development and application of individual Mitochondrial Genome sequencing (iMiGseq) technology.
- Analysis of single human oocytes and a single human blastoid.
- Quantification of SNVs, SVs, and assessment of genetic linkage at the single mtDNA molecule level.
Main Results:
- Identified previously undetected rare heteroplasmic variants in healthy human oocytes, many associated with disease.
- Observed significant shifts in variant frequency and clonal expansion of large SVs during oogenesis.
- Found stable heteroplasmy levels during early lineage differentiation in a human blastoid model.
Conclusions:
- The study provides the first single-mtDNA resolution of heteroplasmy in human oocytes, revealing extensive rare variants.
- Demonstrated dynamic changes in mtDNA heteroplasmy during oogenesis, with implications for offspring health.
- Established a foundation for understanding mtDNA heteroplasmy in early life stages using iMiGseq technology.
