## 3 Results  
Both Restriction Endonucleases Efficiently Cut the Circular Mitochondrial DNA While Simultaneously Fragmenting the Genomic DNA  
In this study, a comparative analysis of PvuII-HF and BamHI-HF was conducted on JAR cell line to evaluate their effectiveness and reliability for mitochondrial genome linearization. This evaluation involved comparing the Qsep-100 fragment profiles of enzyme-treated DNA against an untreated native DNA control (Figure 2). Both enzymes are high-fidelity variants that specifically target their respective recognition sites (CAGCTG for PvuII-HF and GGATCC for BamHI-HF) with minimal star activity. Figure 2 demonstrates that both enzymes successfully linearize the mitochondrial genome, though their target sequences are located at distant positions: BamHI-HF leaves at m.14262, while PvuII-HF cuts at m.2652. PvuII-HF was preferentially selected for further mitochondrial analysis based on two factors: (1) previously reported issues with the GGATCC sequence located within deletion breakpoints affecting coverage profiles [20], and (2) bio-fragment analysis showing longer fragments retained after BamHI-HF treatment (Figure 2). PvuII-HF's site specificity provides consistent and predictable linearization within the mitochondrial genome. An additional consideration is that PvuII-HF has more recognition sites within the nuclear genome (114 times) compared to BamHI-HF, resulting in higher nuclear DNA fragmentation. This characteristic of PvuII-HF provides an advantage for adaptive sampling, where extremely long fragments are less desirable as they may lead to false rejection of targeted sequences. PvuII-HF was subsequently validated on human adipose-derived mesenchymal stem cells (HAdMSCs) using the same fragment analysis comparison to evaluate its efficacy in selectively linearizing mitochondrial DNA while preventing the retention of excessively large DNA fragments. As illustrated in Figure 3, the treatment exhibited high reliability, producing consistent and reproducible fragmentation patterns. This reproducibility underscores PvuII-HF's utility as a precision tool for adaptive sampling studies, where controlling fragment length is critical for accurate analysis. ADME Genes Exhibit Enhanced Coverage Depth and Greater Variant Representation in AS Data Compared to WGS Data  
The integrity of the sequenced ADME genes was not perturbed by the enzyme pre-treatment in both AS and WGS data. The average sequencing coverage depth reported for each ADME gene in both the targeted sequencing and WGS datasets represents a weighted average, calculated by intersecting the defined gene regions with per-base coverage data derived from the respective alignments, multiplying each coverage value by the length of its corresponding genomic segment within the gene, summing these products per gene, and dividing by the total length of segments with coverage data within that gene. Targeted sequencing of the ADME gene panel yielded substantially higher raw read counts and normalized coverage (reads per million mapped reads) for the target regions compared to whole genome sequencing (WGS). The comparison of normalized data revealed relatively uniform sequencing coverage but variable methylation patterns across ADME genes with targeted sequencing, whereas whole genome sequencing showed greater inter-gene variability in normalized coverage alongside similarly variable normalized methylation patterns (Figure 4A, Supplementary S1). Furthermore, ADME genes had slightly better sequencing depth and variant representation in AS compared to WGS data (Figure 4B). This suggests that pure pharmacogenomic profiling could be performed even with AS protocols for the purpose of organ-on-chip studies and pharmacogenomics studies. The analysis of key ADME genes utilizing adaptive sampling (AS) revealed substantially improved sequencing coverage and variant detection compared to standard whole genome sequencing (WGS).