Mitochondrial Communications 2 (2024) 107–113

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Mitochondrial Communications
journal homepage: www.keaipublishing.com/en/journals/mitochondrial-communications

Review Article

Single-cell mitochondrial DNA sequencing: Methodologies and applications

Guoqiang Zhou a,b , Zhenglong Gu b,c,** , Jin Xu d,*
a
HKUST Fok Ying Tung Research Institute, Guangzhou, 511458, China
b
Greater Bay Area Institute of Precision Medicine, Fudan University, Nansha District, Guangzhou, 511400, China
c
School of Life Sciences, Fudan University, Shanghai, 200433, China
d
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, China

A R T I C L E I N F O A B S T R A C T

Keywords: Mitochondria play a pivotal role in cellular energetics, metabolism, and various regulatory processes. Their
Mitochondrial DNA dysregulation is implicated in numerous diseases. Traditional population-level mitochondrial DNA (mtDNA)
Single-cell sequencing sequencing often obscures crucial information from individual cells, leading to a limited understanding of
Mitochondrial heteroplasmy
mitochondrial genetics. In contrast, single-cell mtDNA sequencing enables the precise detection and character­
Mitochondrial genetics
ization of mtDNA mutations at the individual cell level, providing a nuanced view of mitochondrial heteroplasmy
and its dynamics. This review aims to provide a comprehensive overview of current single-cell mtDNA
sequencing methodologies and their applications in advancing our understanding of mitochondrial genetics.

1. Introduction mutant mtDNA frequency exceeds a critical threshold, typically between

60 % and 80 %, the mutation manifests phenotypically.13–15 This
Mitochondria are crucial for the proper functioning of organisms by threshold is not fixed and varies depending on the mutation and tissue
orchestrating a wide array of essential processes, such as ATP generation involved.16 For example, high-frequency mutations in the D-loop region
via oxidative phosphorylation (OXPHOS), maintaining calcium equi­ of the mitochondrial genome can impair mtDNA replication, affecting
librium,1 facilitating iron–sulfur cluster biogenesis,2 regulating cellular energy metabolism.17 Such mutations have also been implicated
apoptosis,3 and coordinating diverse small molecule biosynthesis.4–8 in tumorigenesis.18 Similarly, mutations in mitochondrial tRNA genes
mtDNA is a circular DNA molecule spanning approximately 16–17 kb can disrupt protein synthesis, leading to metabolic dysfunctions and
that is inherited maternally and present in multiple copies per cell. It diseases such as muscle weakness19 and hearing loss.20 In regions
harbors a compendium of 37 genes encompassing 11 mRNA (translated encoding mRNA, high-frequency mutations may result in the mis­
into 13 polypeptides) for OXPHOS, two ribosomal RNA genes, and 22 expression of key subunits of oxidative phosphorylation complexes,
tRNA genes.9 impairing the OXPHOS process, which is particularly relevant in meta­
In contrast to nuclear DNA (nDNA), mtDNA replicates independently bolic disorders and age-related mitochondrial dysfunction.21,22
of the cell cycle. This autonomy in replication coupled with its faster Disruption of the genetic information encoded in mtDNA due to
pace, the lack of several cellular DNA repair mechanisms, and the mutations can lead to malfunctioning of crucial processes of the cell,
absence of histone protection collectively contribute to mutation rates in contributing to the onset of various diseases.23,24 This intricate interplay
mtDNA that are orders of magnitude higher than those in nDNA, ranging between the unique characteristics of mtDNA and its susceptibility to
approximately 10–270 times higher.10 Another unique feature of mutations is crucial for understanding the complexities of mitochondrial
mtDNA is heteroplasmy, whereby mutant mtDNA often coexists with genetics and their consequential impact on health and disease. The
wild-type mtDNA.11,12 Variations in heteroplasmy frequency can lead to predominant approach for detecting mtDNA mutations involves
distinct phenotypic outcomes depending on the specific mutation. This population-level sequencing, which is valued for its experimental
relationship is governed by the phenotypic threshold effect; that is, at simplicity and cost-effectiveness. This method is effective at determining
low heteroplasmy levels, the harmful effects of mutant mtDNA are often the average mutation frequencies across cell populations; however, it
masked by the presence of wild-type mtDNA copies. However, once the overlooks the variation in mutation frequencies across individual cells.25

* Corresponding author.
** Corresponding author. Greater Bay Area Institute of Precision Medicine, Fudan University, Nansha District, Guangzhou, 511400, China.
E-mail addresses: guzhenglong@ipm-gba.org.cn (Z. Gu), xujin7@mail.sysu.edu.cn (J. Xu).

https://doi.org/10.1016/j.mitoco.2024.10.001
Received 22 August 2024; Received in revised form 21 October 2024; Accepted 30 October 2024
Available online 30 October 2024
2590-2792/© 2024 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
G. Zhou et al. Mitochondrial Communications 2 (2024) 107–113

Fig. 1 illustrates two cell populations with an identical mean variant combinatorial indexing, adapted from sci-RNA-seq27 and SPLiT-seq,28 to
allele frequency (VAF) of 0.25, yet their variation and functional im­ capture mitochondrial transcripts. This significantly enhances scalabil­
pacts may differ significantly. In the first group, a quarter of the cells ity and reduces sequencing costs.
exhibit a homoplasmic mutation, while the remaining cells exclusively The fundamental principle of droplet-based microfluidics involves
harbor wild-type mtDNA. Conversely, the second cell population con­ encapsulating individual cells within tiny droplets of a water-in-oil
sists of 100 % of cells, with an average of 25 % heteroplasmy. Despite emulsion, a technique commonly employed by platforms such as those
both scenarios presenting an apparent VAF of 0.25, the functional con­ developed by 10X Genomics.29 These droplets function as isolated re­
sequences at the tissue level can be markedly different. The first popu­ action chambers, enabling a variety of biochemical reactions. By uti­
lation, characterized by the localized presence of homoplasmic lizing microfluidic channels, this method partitions samples into
mutations, may lead to pronounced tissue effects. In contrast, the second droplets, allowing for high throughput through parallel processing of
population, marked by the widespread distribution of heteroplasmy multiple samples simultaneously. Single-cell mtDNA sequencing tech­
across all cells, elicits a more subtle impact on tissue function. Therefore, nologies based on droplet-based microfluidics include MAESTER,30
precise detection of mtDNA mutations requires single-cell resolution, mtscATAC-seq,31,32 DOGMA-seq,33 and ReDeeM.34 Compared to
providing unparalleled insights into functional dynamics. Recent ad­ well-based methods, droplet-based approaches offer significantly higher
vancements in single-cell omics technologies have made precise mea­ throughput. Moreover, the small volume of each droplet, typically at the
surement of mtDNA mutations at the single-cell level feasible. This picoliter (pL) or nanoliter (nL) scale, minimizes reagent consumption,
review strategically summarizes the latest methodologies in single-cell thereby reducing the experimental costs per cell. However, these
mtDNA sequencing and their applications across diverse research areas. methods present challenges, such as the complexities of microfluidic
handling and the need for specialized equipment and requisite expertise,
2. Single-cell methods for mtDNA sequencing which may limit accessibility for researchers without specific resources
or training. Table 1 provides an overview of current single-cell mtDNA
Despite variations in experimental procedures, protocols for single- sequencing methods, categorized by platform and corresponding
cell mtDNA sequencing generally involve three key steps: cell isola­ throughput per run.
tion, mtDNA amplification or direct capture for library preparation, and
sequencing. Variability at each stage can significantly impact the 2.2. Strategies for constructing an mtDNA library
detection of mtDNA mutations at the single-cell level. In this section, we
systematically compile and compare the latest advancements in single- MtDNA amplification is crucial in cases with very limited sample
cell mtDNA sequencing, offering a comprehensive understanding of amounts, such as single-cell experiments, as it increases detectable DNA
technical intricacies to guide informed decisions across diverse levels, ensuring successful analysis. Additionally, amplification en­
applications. hances the detection of low-frequency mutations, particularly when
mtDNA mutation frequencies are extremely low, by amplifying rare
2.1. Cell separation for single-cell mtDNA sequencing mutation signals and improving sensitivity. In this section, we delve into
the specific protocols employed by different methods to amplify or
Single-cell mtDNA sequencing approaches can be broadly classified enrich the mtDNA genome and examine their impact on mutation
into two categories based on cell isolation methods: well-based plat­ quantification. Generally, strategies for mtDNA amplification or
forms and droplet-based microfluidics. Well-based platforms isolate in­ enrichment are more commonly applied within well-based platforms,
dividual cells via flow cytometry, placing them into separate wells on owing to the open and easily manageable nature of microplates.
microplates (e.g., 96-well or 384-well plates). Each well is then inde­ The first single-cell mtDNA sequencing method was developed spe­
pendently processed for mtDNA amplification and dual indexing, ac­ cifically for oocytes, exploiting their high mtDNA copy number.38,39
commodating a variety of sequencing techniques tailored to specific This approach employs four PCR primers to amplify the entire mito­
research objectives, including studies on oocytes and other cell types. chondrial genome, covering regions 3561–9794, 9795–14,567, 14,
The adaptability of this platform arises from its open configuration, 562–139, and 115–3560. Following standard Illumina library prepara­
which allows for multistep reactions and buffer exchanges and mini­ tion and sequencing, this method, known as Oocyte-seq, enables precise
mizes cross-contamination between cells. However, well-based methods mutation detection in single oocytes.35 In contrast to oocytes, which
generally suffer from low throughput, labor-intensive operations, and have a high mtDNA copy number, the amplification of mtDNA from
high costs per cell. A recent innovation, SCI-LITE,26 offers a single somatic cells with lower copy numbers presents significant chal­
high-throughput alternative by employing split-pool barcoding and lenges, particularly with long-range amplification methods. To over­
come these challenges, Ludwig et al. introduced rolling-circle
amplification (RCA) for mtDNA amplification in single somatic cells.37
This method enriches full-length circular mtDNA using custom
mtDNA-specific primers or a commercially available kit, effectively
reducing contamination from nuclear-encoded mtDNA. RCA leverages
the enzymatic activity of Phi29 polymerase for isothermal amplification,

Table 1
Comparison of Single-Cell mtDNA Sequencing Technologies Based on Well and
Droplet Platforms.
Method Platform Throughput (cells per run)
35
Oocyte-seq Well 96–384
scSTAMP36 Well 96–384
scMito-seq37 Well 96–384
SCI-LITE26 Well 10000–100000
MAESTER30 Droplet 10000–20000
mtscATAC-seq31,32 Droplet 10000–20000
DOGMA-seq33 Droplet 10000–20000
Fig. 1. Bulk Analysis of mtDNA Mutations Falls Short in Discerning Diverse
ReDeeM34 Droplet 10000–20000
Situations with Varying Physiological Outcomes.

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achieving a lower error rate and ensuring the accurate identification of Table 2
single nucleotide variations. By integrating RCA with Tn5 tagmentation Comparison of Single-Cell mtDNA Sequencing Technologies Based on mtDNA
for library construction, scMito-seq was developed, facilitating mtDNA Amplification/Enrichment and Nonamplification/Nonenrichment. The
sequencing in single somatic cells and allowing for variant coverage of mtDNA is defined as the average sequencing depth per cell. “Low”
quantification.37 coverage is characterized by a mean depth between 0.5X and 10X, “medium”
coverage denotes a mean depth between 10X and 100X, and “high” coverage
PCR-based mtDNA amplification for library construction is widely
refers to a mean depth greater than 100X.
favored for its cost-effectiveness and efficiency. However, it may intro­
duce false-positive mutations. To mitigate this issue, the scSTAMP Method Strategy mtDNA- UMI Coverage of
specific mtDNA
method36,40 was developed. Initially, scSTAMP performs long-range
primers
amplification of two segments (encompassing regions 2933–11368
scATAC-seq41 Tn5 transposition No No Low
and 11299–2958) to cover the entire mtDNA. The amplified products
scRNA-seq48 PolyT barcoded No Yes Low
are then captured using single-stranded oligonucleotide probes through primer capture
an extension–ligation reaction. The extension probe features a 3′ Oocyte-seq35 mtDNA amplification Yes No High
extension arm complementary to the mtDNA target, a unique molecular scSTAMP36 mtDNA amplification Yes Yes High
tag for tracking the capture event, and a PCR primer annealing region for scMito-seq37 mtDNA amplification Yes No High
SCI-LITE26 mtRNA RT with Yes Yes Median
subsequent amplification. The ligation probe includes a 5′ phosphory­
barcoded primer
lated arm complementary to the mtDNA target and a 20-nt PCR primer mtscATAC- Tn5 transposition No No Median
annealing region at its 3′ end. These probes selectively amplify the seq31,32
mtDNA parental strand, with the resulting products carrying unique DOGMA- Tn5 transposition No Yes Median
seq33
molecular identifiers (UMIs). scSTAMP enhances the accuracy of mtDNA
MAESTER- mtcDNA PCR-based Yes Yes High
mutation detection by minimizing PCR errors. seq30 enrichment
In contrast to well-based methods, droplet-based approaches often ReDeeM34 mtDNA specific Yes Yes High
involve the direct capture or enrichment of mtDNA following major li­ probes-based capture
brary construction steps. For instance, standard protocols in single-cell
RNA and single-nuclei ATAC sequencing directly capture mitochon­
molecule sequence capabilities for mtDNA.
drial transcripts and genomes. However, these methods frequently suffer
In NGS methodologies, DNA is typically fragmented into smaller
from uneven mtDNA coverage and limited sequencing depth, hampering
pieces prior to sequencing, facilitating efficient and high-throughput
their ability to detect and quantify mtDNA mutations effectively.
analysis. However, conventional NGS-based approaches face chal­
To address these limitations, new protocols have been developed to
lenges when detecting rare heteroplasmic variants with allele fre­
improve mitochondrial sequence capture or enrichment. MAESTER,30
quencies below 1 %. This difficulty arises from the background error
for example, enhances the capture of full-length cDNA transcripts in
rates associated with NGS, which range from 0.1 % to 1 %.49,50 These
single-cell 3′ transcriptome sequencing using the 10X Genomics kit.
error rates, resulting from the inaccuracies introduced during
During this process, mitochondrial transcripts are selectively enriched
sequencing, base calling, PCR amplification, and library preparation,
with pools of primers while preserving cell-identifying barcodes. This is
can obscure low-frequency variants, complicating accurate detection
followed by standard next-generation sequencing with 250 bp reads,
and quantification.
resulting in more than a 50-fold increase in mitochondrial transcriptome
TGS platforms, such as those from PacBio and Oxford Nanopore
coverage and robust mutation detection.
Technologies, have transformed mtDNA analysis by enabling the
With the single-cell ATAC sequencing protocol,41 researchers have
sequencing of long DNA fragments. This capability allows for the cap­
employed mild cell lysis or permeabilization instead of nuclear isolation,
ture of full-length mtDNA sequences, which are essential for detailed
allowing the Tn5 enzyme to integrate adapters into both accessible
variant characterization and complete haplotype resolution. A signifi­
nuclear chromatin and mtDNA. This method, known as mtscA­
cant advancement in this field is the use of UMIs to tag individual
TAC-seq,31,42,43 increases mtDNA capture 361-fold. Expanding on this
mtDNA molecules within single cells. After UMI tagging, high-fidelity
approach, Mimitou et al. developed ASAP-seq33,44 by integrating
long-range PCR amplification is performed, and the amplified prod­
oligonucleotide-labeled antibodies to capture cellular proteins and
ucts are sequenced using long-read platforms. This approach addresses
optimizing fixation conditions to modulate cell surface labeling and
the limitations of conventional NGS, enhancing both the accuracy and
retain mtDNA. This technique, in combination with the 10X multiome
depth of mtDNA sequencing. The ability to obtain full-length mtDNA
kit, known as DOGMA-seq,33 excels in comprehensive single-cell anal­
sequences and resolve individual haplotypes is particularly valuable for
ysis, enabling simultaneous profiling of cell surface proteins and mtDNA
studying mitochondrial heterogeneity and inheritance patterns. TGS
mutations as well as transcriptome and chromatin accessibility.45,46
provides several advantages over NGS, including fewer assembly errors
Further enhancing mtDNA enrichment, Weng et al. developed
and enhanced detection of structural variations (Fig. 2). Technologies
ReDeeM,47 which, in addition to the ASAP-seq protocol utilizing cell
such as single-cell mtDNA long-fragment amplification, exemplified by
barcoding beads for capturing both Nextera capture sequences and
scMito-seq, can be seamlessly integrated with TGS platforms. This
polyA sequences, employs specially designed mtDNA probes to enrich
integration enables a comprehensive analysis of mtDNA at the single-
mtDNA molecules post-demulsification. This method significantly im­
cell level, advancing our understanding of mitochondrial function, dis­
proves sequencing depth, enabling a comprehensive analysis of mtDNA
ease, and evolution.
mutations at the single-cell level. Table 2 summarizes the discussed
methods, their respective strategies for mtDNA readout, and their effects
3. Applications of single-cell mtDNA sequencing
on mtDNA enrichment.

3.1. Applications for advancing the functional effects of mtDNA

2.3. Sequencing platform for mitochondrial genome readout
mutations

Next-generation sequencing (NGS) and third-generation sequencing

Most regions of the mitochondrial genome are essential for funda­
(TGS) platforms are utilized for mitochondrial genome analysis. NGS
mental mitochondrial functions. Variations in mtDNA heteroplasmy
remains the predominant strategy due to its widespread application and
levels can result in distinct cellular phenotypes (Fig. 3A). Bulk mtDNA
well-established protocols. It offers distinct advantages, particularly in
sequencing may erroneously suggest that low-frequency mtDNA
capturing linkage and haplotype information, and provides single-

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Fig. 2. Advantages of TGS in Haplotyping.

Fig. 3. Functional Effects and Genetic Dynamics of mtDNA Variants.

mutations lack functional significance. The advent of single-cell mtDNA copies, regardless of the mutation’s effect.22 In this context, the mtDNA
sequencing has introduced a high-resolution method for detecting low- copy number is analogous to the population size, determining the in­
frequency mtDNA mutations, thereby providing valuable insights into fluence of random genetic drift. Notably, unlike in a single or limited
the biological roles of mtDNA. For instance, Yang et al. demonstrated number of natural populations, the variant allele frequency of an mtDNA
that mtDNA mutations accumulated with aging are inversely correlated mutation can be observed across a larger number of descendant cells. A
with female fertility. Mechanistically, these mutations impair the lower mtDNA copy number in ancestry cells leads to a greater variance
NADH/NAD+ redox state, leading to a reduction in ovarian primordial in heteroplasmy among descent cells. Consequently, the variance in
and mature follicles.35 Similarly, Guo et al. uncovered the pervasive and heteroplasmy frequency among cells serves as a metric for assessing the
notably detrimental presence of mtDNA mutations at the single-cell impact of an mtDNA genetic bottleneck.
level in aging individuals.36 Single-cell mtDNA sequencing has also A notable phenomenon is the genetic bottleneck observed during
elucidated the dynamics of pathogenic mtDNA heteroplasmy, revealing oocyte development and early embryogenesis,52 which significantly
patterns consistent with purifying selection and distinct metabolic vul­ reduces the mtDNA copy number. This bottleneck likely serves as a
nerabilities across T-cell states.45 Additionally, this technology has quality control mechanism by filtering out deleterious mutations to
proven effective in identifying skewed immune-cell expansions in pri­ prevent their accumulation.53 Similarly, single-cell mtDNA sequencing
mary human clonal hematopoiesis.33 has revealed that somatic cells also experience genetic bottlenecks, as
evidenced by an overrepresentation of somatic homoplasmic mtDNA
mutations in lymphocytes,54 suggesting that similar mtDNA quality
3.2. Applications for advancing the understanding of mitochondrial control mechanisms may be present in somatic cells.
genetics The variant copy number of mtDNA molecules across different cell
types not only correlates with metabolic demands but also affects the
Somatic mtDNA mutations often manifest as heteroplasmy, whereby dynamics of mtDNA variants and genotypes, potentially altering cellular
the frequency of mutant alleles significantly impacts mitochondrial functions. Several studies have highlighted that mtDNA variants can be
dysfunction in individual cells. Understanding the variation in hetero­ stable or accumulate over time, correlating with age-related cellular
plasmy among different cells and identifying factors that influence these dysfunction. For example, research on various mouse tissues has indi­
dynamics are central to mitochondrial genetics in the era of single-cell cated an age-dependent accumulation of somatic homoplasmic muta­
genomics. tions.55 Similarly, mtDNA variants in chronic lymphocytic leukemia
Random genetic drift is an evolutionary mechanism that leads to have shown stability over years, illustrating a complex interaction be­
changes in allele frequencies within a population due to random sam­ tween disease progression and mtDNA dynamics.56 Additionally,
pling effects.51 This process can lead to the loss of genetic variation and induced pluripotent stem cells derived from older individuals may
may result in certain alleles becoming fixed in the population over harbor significant amounts of mtDNA mutations.57 In summary, the
generations. The impact of genetic drift is particularly pronounced in effects of genetic drift on mtDNA vary among cell types and accumulate
small populations in which stochastic events can have a more substantial over time or generations.
effect on allele frequencies. The same stochastic process influences In addition to random genetic drift, mtDNA mutation dynamics are
mtDNA mutation frequency within individual cells (Fig. 3B). When a influenced by cellular selection processes. Studies on pathogenic mtDNA
new mutation arises in a single mtDNA molecule, it can undergo random variants have shown cell type–specific selection. For instance, Walker
drift, resulting in either an increase or decrease in the number of mutant

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et al. observed a significant reduction in the heteroplasmy of the 3243A Single-cell multi-omics sequencing applications for lineage tracing
> G mutation in T cells from patients with mitochondrial encephalo­ have been extensively demonstrated in hematopoietic cells and cancer
myopathy, lactic acidosis, and stroke-like episodes, suggesting purifying cells.69 Utilizing somatic mtDNA mutations as natural genetic barcodes
selection within lymphocyte lineages.58 This purifying selection of is a promising strategy for resolving cell lineage relationships in human
pathogenic mtDNA variants has been corroborated by other studies.59–61 tissues. However, careful consideration of the genetic dynamics of
Franklin et al. further reported a decline in variant allele frequency and a mtDNA variants, as discussed earlier, is essential for the accuracy of
decrease in mtDNA copy number with age in T cells from mitochondrial inferred lineage relationships. Benchmarking lineage relationships
disease patients.59 Notably, a lineage-specific genetic bottleneck in through whole-genome single-cell sequencing70 and computational
lymphocytes may enhance cell type–specific purifying selection.54 Se­ simulations71 has provided practical methods and emphasized the
lection strength varies based on the functional requirements of different importance of evaluating lineage relationships inferred from mtDNA
cell types and can also depend on environmental conditions. For mutations (Fig. 4).
example, a recent study demonstrated that the same mtDNA variant
could be harmful, neutral, or beneficial depending on the culture envi­ 4.2. mtDNA variants as intrinsic barcodes for sample demultiplexing
ronment.26 This suggests that distinct metabolic requirements may drive
mtDNA variant dynamics. Pooling cells from multiple individuals offers significant advantages
Beyond the genetic bottleneck and purifying selection at the cellular for cohort studies, including increased sample size, reduced single-cell
level, the cis effects of genetic variants also influence mitochondrial sequencing costs, and minimized batch effects.45 Demultiplexing
DNA amplification and quality control, further contributing to their own mixed samples in single-cell sequencing can be accomplished through
dynamics.22,25 In some cases, mutated mtDNA may replicate more either exogenous or endogenous labeling methods.72–74 Exogenous la­
efficiently than wild-type mtDNA, leading to an increased proportion of beling typically employs antibodies conjugated with nucleotide se­
mutated mtDNA (Fig. 3C). For instance, Kim et al. found that the quences. However, the specificity of current “universal antibodies” is
m.302A heteroplasmy was associated with a higher mtDNA copy num­ often inadequate, leading to the loss of certain cell types.75 Furthermore,
ber, indicating that the mtDNA genotype itself may modulate mtDNA the labeling process involves repetitive centrifugation and washing
copy number levels. Conversely, some mutations may result in less steps, which can compromise cell viability. Endogenous labeling
efficient replication, decreasing their relative proportion over time. methods, in contrast, utilize genetic variants between individuals for
Additionally, the mitochondrial quality control system, including sample demultiplexing. While single-nucleotide polymorphisms in the
mitophagy (the selective degradation of damaged mitochondria), can nuclear genome can be challenging due to high dropout rates, mito­
influence the frequency of mtDNA mutations. When mutations lead to chondrial variants exhibit a lower dropout rate in single cells, making
dysfunctional mitochondria, quality control mechanisms may be trig­ them efficient endogenous barcodes. This approach allows for the pre­
gered, resulting in the selective degradation of mitochondria containing cise demultiplexing of high-throughput single-cell mixed samples.76,77
defective mtDNA. This process can affect the overall mtDNA copy Endogenous labeling not only enhances the accuracy of single-cell
number and heteroplasmy frequency within cells. sequencing data but also overcomes the limitations of current labeling
In summary, advances in single-cell mtDNA sequencing have pro­ methods, offering a cost-effective and robust solution for demultiplexing
vided new insights into the dynamics of mtDNA heteroplasmy, revealing single-cell samples (Fig. 4).
complex cell type–specific mechanisms in mitochondrial genome in­
heritance. Observations from recent studies suggest that mtDNA variant 4.3. Detection of cellular exchanges of mitochondria via mtDNA variants
dynamics vary across cell types and over time and are influenced by
genetic drift, selection, environmental interactions, and the cis effects of Research into intercellular mitochondrial transfer has revealed its
variants. Further exploration across broader spatial and temporal di­ critical role in both normal physiology and disease states78 (Fig. 4). For
mensions will deepen our understanding of mitochondrial inheritance example, mitochondria can transfer unidirectionally from T cells to
mechanisms, offering insights into mitochondrial diseases and aging. cancer cells, leading to reduced T cell immune activity and enhanced
metabolic capabilities in cancer cells, which facilitate immune
4. Application of mtDNA variants as genetic markers for evasion.79 This phenomenon has been demonstrated across various
individual cells cancer types using fluorescence-labeled mitochondria from
KrasG12D/p53ko cancer cells cocultured with CD8+ T cells.80 A ma­
4.1. Cell lineage tracing using mtDNA variants as genetic markers chine learning algorithm, MERCI, was developed to track mitochondrial
transfer at single-cell resolution using single-cell transcriptomic data.
Understanding the lineage relationships among cells is crucial for MERCI revealed the prevalence of mitochondrial transfer and its asso­
studying normal development and disease progression. While various ciation with the TNF-α pathway in human tumor samples.80 Addition­
recording systems have been developed for constructing cell lineages in ally, mitochondrial transfer has been found to correlate with increased
model organisms, these methods are not directly applicable to humans. cell cycle activity in cancer cells and poorer clinical outcomes,80 high­
Retrospective lineage tracing using endogenous genetic markers offers lighting its significance in cancer biology and its potential implications
an alternative solution. However, many current methods rely on for immunotherapy development. Despite these advancements, our
detecting nuclear somatic mutations, which face challenges such as high understanding of the molecular mechanisms underlying various inter­
error rates, limited scalability, and insufficient capture of critical cell cellular mitochondrial transfer pathways remains limited. These mech­
state information.62–65 anisms are likely specific to the cell type, tissue, and context. Further
MtDNA variants, due to their high mutation rate and ease of readout, elucidation of these pathways could revolutionize our understanding of
present an ideal class of endogenous genetic markers for lineage tracing. mitochondrial biology and open new avenues for therapeutic
The concept of using mtDNA mutations for lineage tracing was proposed intervention.
before the advent of single-cell genomics.66,67 Recent research has
demonstrated that somatic mtDNA mutations, when effectively read out, 5. Challenges and future directions
can be used to construct lineage relationships among cells.37,68 This
approach has been validated in human immune cells, highlighting the In summary, single-cell mtDNA sequencing has revolutionized our
potential of mtDNA variants as natural genetic barcodes for lineage ability to explore mtDNA variation at the level of individual cells,
tracing. This has spurred further research into optimizing methods for shedding light on its contribution to cellular function. Despite these
simultaneously capturing mtDNA and cell fate information.32,33 significant advances, further innovations are required to overcome

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G. Zhou et al. Mitochondrial Communications 2 (2024) 107–113

Fig. 4. Application of mtDNA variants as genetic markers.

ongoing challenges. health and disease. We anticipate that future innovations will usher in a
First, optimizing the balance between throughput and mtDNA new era of mtDNA sequencing technologies, providing unprecedented
coverage is crucial. Well-based single-cell mtDNA sequencing offers high insights into mitochondrial genetics.
coverage and sequencing depth; however, it suffers from low throughput
and lacks the capability to concurrently sequence other omics. CRediT authorship contribution statement
Conversely, droplet-based techniques facilitate high-throughput and
cost-effective single-cell mtDNA sequencing and enable simultaneous Guoqiang Zhou: Writing – original draft, Visualization. Zhenglong
analysis with other omics, although they generally provide a lower Gu: Writing – review & editing, Validation, Supervision, Conceptuali­
sequencing depth. To address these limitations, there is a pressing need zation. Jin Xu: Writing – review & editing, Visualization, Validation,
for the development of advanced sequencing methods that combine high Supervision, Conceptualization.
throughput and high sequencing depth with the simultaneous analysis of
mtDNA and multi-omics. This may involve integrating mtDNA amplifi­
cation steps into existing single-cell multi-omics platforms, such as the Declaration of competing interest
HetHydroge81 platform. Cost-effective technologies will also play a
crucial role in facilitating large-scale studies, which are essential for I certify that this is an original manuscript and that none of the
elucidating the pivotal role of mtDNA in mitochondrial dysfunc­ material has been published or is under consideration elsewhere,
tion–related disorders. including the internet. All authors have agreed to all the content in the
Second, multi-omics readouts at the single-cell level will be critical manuscript and none of them has any competing financial interest.
for understanding the functional consequences of mtDNA variation.
Previous studies have shown that the dysregulation of mitochondria Acknowledgments
function significantly alters metabolism in cells, which in turn affects
nuclear epigenetics. For instance, a progressive increase in human This work was supported by the National Key R&D Program of China
mtDNA heteroplasmy can lead to distinct nuclear epigenomic changes (2021YFA1102100 to J.X.), the National Natural Science Foundation of
by affecting nuclear histone acetylation, thereby inducing transcrip­ China (32070644, 32293190, and 32293191 to J. X., 32293210 to Z.G.),
tional reprogramming of the nuclear genome.82,83 Additionally, a the Guangzhou Basic and Applied Basic Research Special Program
complex relationship exists between the mtDNA copy number and the (SL2022A04J00717 to G.Z., 2024A1515011285 to J.X.), and the
global nuclear DNA methylation level.84These findings underscore the Guangzhou Key Research and Development Program (2023B03J1347 to
need for technologies that can simultaneously read out mtDNA genotype J.X).
and nuclear epigenetic information. In addition, integrating TGS with
high-throughput single-cell platforms allows for the capture of haplo­ References
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