## 3 Results
\begin{table}
\begin{tabular}{c c c c} \hline
**Haplotype ID** & **Frequency (\%)** & **Read Count** & **Major Variant Positions** \\ \hline Haplotype 1 & 3.6 & Highest & 310 T\textgreater{}C, 625 G\textgreater{}A variants; conserved blue regions at 308-309 C\textgreater{}T \\ \hline Haplotype 2 & 2.2 & High & Conserved blue regions at 308-309 C\textgreater{}T; red variant at 2001 C\textgreater{}T \\ \hline Haplotype 3 & 2.2 & High & Distinctive red variants at 625 G\textgreater{}A and 2001 C\textgreater{}T \\ \hline Haplotype 4 & 1.5 & Medium & Red variant at 2001 C\textgreater{}T; missing blue variants at 308-309 \\ \hline Haplotype 5 & 1.5 & Medium & 625 G\textgreater{}A variant; missing variants at 2001 and 2230 \\ \hline Haplotype 6 & 1.5 & Medium & 625 G\textgreater{}A and 2230 A\textgreater{}G variants \\ \hline Haplotype 7 & 1.5 & Medium & 2001 C\textgreater{}T variant; missing the common 308-309 blue pattern \\ \hline Haplotypes 8–63 & 0.7 each & Low & Various combinations of variants with low frequency \\ \hline \end{tabular}
\end{table} 
Table 6: Summary of major mtDNA haplotypes using WGS data. Analysis of the variant structure (Figure 9D) reveals several conserved variant patterns across the major haplotypes. The C\(>\)T variants at positions 308-309 are ubiquitous in Haplotypes 1-7, appearing as a conserved blue region, which may represent a founder mutation in this cell line. Other significant variants include G\(>\)A at position 625 (prominent in Haplotypes 1, 3, 5, and 6) and C\(>\)T at position 2001 (common in Haplotypes 2, 3, 4, and 7). The A\(>\)G variant at position 2230 appears selectively in Haplotypes 2 and 6, while the A\(>\)G variant at position 1347 and C\(>\)T at position 1428 show variable distribution across the haplotypes. Compared to the previous adaptive sampling (AS) sequencing analysis, this WGS approach has revealed a substantially more complex mtDNA landscape, with greater haplotype diversity and more nuanced clustering patterns. While the major relationship patterns are consistent between both analyses (e.g., the close relationship between Haplotypes 2 and 5), the WGS data provide a more comprehensive view of the mitochondrial genetic structure in this MSC primary cell line, capturing rare variants and subtle relationships that were not detected in the more targeted AS approach. The hierarchical clustering of mtDNA haplotypes directly informed our haplogroup classification, with dominant haplotypes from both sequencing approaches converging  
Figure 9: Mitochondrial DNA haplotype diversity analysis in MSC primary cell line. **(A)** Hierarchical clustering dendrogram of 63 mtDNA haplotypes based on genetic distances, revealing multiple distinct lineage clusters. **(B)** Haplotype distance matrix showing pairwise genetic distances between all mtDNA haplotypes, with colors ranging from yellow (identical, 0.0) to dark blue (maximum difference, 1.0). **(C)** Multidimensional scaling plot illustrating relative genetic relationships between haplotypes in two-dimensional space, with dot coloring indicating haplotype frequency. **(D)** Variant patterns across key nucleotide positions for 15 selected haplotypes (with frequencies shown in parentheses), where red indicates one allelic variant, blue indicates the alternative allele, and gray represents missing data or intermediate values. on haplogroup assignment T2b28 for AdMSC cells, providing a genetic context for interpreting the functional characteristics of these cell lines in our organ-on-chip models (Supplementary Figures S2-S5). Direct Nanopore Sequencing Reveals Low Non-CpG Methylation Levels in Mitochondrial DNA Independent of Sequencing Coverage  
Leveraging the ability of nanopore sequencing to detect base modifications directly on native DNA strands, we investigated cytosine methylation patterns within the mitochondrial genome (mtDNA) of HAdMSCs.