Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA (mtDNA) Variant Detection, with an Emphasis on Detection and Quantification of mtDNA Deletion

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a highly technical article comparing long-read sequencing (LRS) versus short-read sequencing in mtDNA. The authors have demonstrated that LRS has the potential to be introduced as a clinical test to replace conventional techniques.

However, we have had to make an effort to properly understand Table 1. We remind the authors that a table should be interpretable on its own. Therefore, we recommend that they indicate in the footnote what S1, S2…, M1…, and other abbreviations mean, so that it can be understood without having to refer back to the main text.

Author Response

Comments 1: This is a highly technical article comparing long-read sequencing (LRS) versus short-read sequencing in mtDNA. The authors have demonstrated that LRS has the potential to be introduced as a clinical test to replace conventional techniques.

However, we have had to make an effort to properly understand Table 1. We remind the authors that a table should be interpretable on its own. Therefore, we recommend that they indicate in the footnote what S1, S2…, M1…, and other abbreviations mean, so that it can be understood without having to refer back to the main text.

Response 1: Thank you for the suggestion. We’ve added table notes at page 4 line 99-104, specified sample types, and spelled out the abbreviations for MMD and LRS. Additionally, we’ve changed RNR2 to MT-RNR2 gene in table head. 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript evaluates PacBio HiFi long-read sequencing (LRS) for comprehensive mtDNA variant detection (SNVs/indels and large-scale deletions) and heteroplasmy estimation, benchmarking against a clinical long-range PCR + short-read sequencing (SRS) workflow and ddPCR. Using 17 samples (SLSMD, MMD, and controls), the authors report high concordance for SNVs >10% heteroplasmy, accurate breakpoint resolution, and strong correlation between LRS-derived deletion heteroplasmy and ddPCR (reported r² ≈ 0.95). The study addresses an important diagnostic gap - accurate breakpoint mapping and deletion heteroplasmy quantification without amplification bias - and suggests LRS could be valuable for complex mtDNA deletion cases.

 

There are a few major concerns here:

1. Clarify the clinical claim boundaries: sensitivity limitations for low heteroplasmy may materially affect conclusions. The manuscript repeatedly indicates decreased sensitivity below ~10% heteroplasmy (for deletions and for SNVs with the current caller settings), which should be foregrounded in the abstract/conclusion and framed as a key limitation to avoid over-generalization. The authors can add a prominent statement (Abstract + Discussion) specifying validated detection threshold(s) for deletions and SNVs and tissue-specific constraints (blood vs muscle depth differences); and provide a sensitivity analysis (or at least descriptive) showing detection probability vs depth/heteroplasmy using your downsampling experiment.

 

2. Strengthen heteroplasmy agreement analysis beyond correlation. Correlation (r/r²) is not sufficient to demonstrate agreement, especially for clinical reporting where bias matters. Please add Bland–Altman plots (LRS vs ddPCR for SLSMD; consider separate for MMD) and report mean bias + limits of agreement; and report absolute error (e.g., median |Δheteroplasmy|) and clinically relevant thresholds (e.g., within ±5% and ±10%).

 

3. Define the “ground truth” comparator for deletions more rigorously, especially in MMD

For MMD, ddPCR is acknowledged to be imperfect because probe regions may be included/excluded variably depending on breakpoints, which complicates using ddPCR as a reference. Please make explicit what constitutes “truth” for MMD: ddPCR, SRS detection, and/or breakpoint-consistent LRS evidence. Where ddPCR is limited, consider orthogonal validation for a subset (e.g., Southern blot, long-range PCR breakpoint-specific assays, or targeted qPCR across multiple loci).

 

4. Sample selection and cohort size: address representativeness and potential spectrum bias

The cohort includes only 17 samples with specific case-mix (4 SLSMD, 9 MMD muscle, 4 controls), including replicates for two samples. This is reasonable for a feasibility study, but the paper should better justify representativeness and how performance may vary across typical clinical samples and tissues.

Please add clearer inclusion criteria and whether samples were consecutive vs convenience-selected. Discuss expected performance in low-mtDNA-content samples (blood) and mtDNA depletion contexts.

Author Response

Comments 1: Clarify the clinical claim boundaries: sensitivity limitations for low heteroplasmy may materially affect conclusions. The manuscript repeatedly indicates decreased sensitivity below ~10% heteroplasmy (for deletions and for SNVs with the current caller settings), which should be foregrounded in the abstract/conclusion and framed as a key limitation to avoid over-generalization. The authors can add a prominent statement (Abstract + Discussion) specifying validated detection threshold(s) for deletions and SNVs and tissue-specific constraints (blood vs muscle depth differences); and provide a sensitivity analysis (or at least descriptive) showing detection probability vs depth/heteroplasmy using your downsampling experiment.

Response 1: We appreciate the reviewer’s suggestion. We’ve added a more detailed descriptive sensitivity analysis based on our downsampling experiment to the manuscript results page 7, line 217-226. Additionally, we’ve made edits to the abstract (page 1, line 26-30).

Comments 2: Strengthen heteroplasmy agreement analysis beyond correlation. Correlation (r/r²) is not sufficient to demonstrate agreement, especially for clinical reporting where bias matters. Please add Bland–Altman plots (LRS vs ddPCR for SLSMD; consider separate for MMD) and report mean bias + limits of agreement; and report absolute error (e.g., median |Δheteroplasmy|) and clinically relevant thresholds (e.g., within ±5% and ±10%).

Response 2: Thanks for the suggestion. We’ve created separate Bland-Altman plots for SLSMD and MMD, which were added as Supplementary Figure 2. Additionally, we’ve included the analysis in the results section (Page 5, line 148-158).

Comments 3: Define the “ground truth” comparator for deletions more rigorously, especially in MMD. For MMD, ddPCR is acknowledged to be imperfect because probe regions may be included/excluded variably depending on breakpoints, which complicates using ddPCR as a reference. Please make explicit what constitutes “truth” for MMD: ddPCR, SRS detection, and/or breakpoint-consistent LRS evidence. Where ddPCR is limited, consider orthogonal validation for a subset (e.g., Southern blot, long-range PCR breakpoint-specific assays, or targeted qPCR across multiple loci).

Response 3: We appreciate the reviewer’s suggestion. We’ve added the detection limits of other orthogonal methods (Southern blot, array CGH, and MLPA) to the discussion (page 10, line 312-316). We’ve also indicated that LR-PCR-based NGS is the most sensitive method for detecting mtDNA deletions. Breakpoint-specific PCR can capture some of the deletion molecules in MMD, but it can’t provide accurate heteroplasmy quantification based on current technology. Other qPCR methods may not exceed the detection sensitivity of ddPCR. We’re working on finding a better method to quantify mtDNA deletions at very low levels.

Comments 4: Sample selection and cohort size: address representativeness and potential spectrum bias. The cohort includes only 17 samples with specific case-mix (4 SLSMD, 9 MMD muscle, 4 controls), including replicates for two samples. This is reasonable for a feasibility study, but the paper should better justify representativeness and how performance may vary across typical clinical samples and tissues. Please add clearer inclusion criteria and whether samples were consecutive vs convenience-selected. Discuss expected performance in low-mtDNA-content samples (blood) and mtDNA depletion contexts.

Response 4: We appreciate the reviewer’s suggestion. We’ve added detailed sample inclusion and exclusion criteria to “4.1 Cohort and data collection,” page 12, lines 422-425 and 429-430. Additionally, we’ve included a discussion of the expected SNV detection in samples with the lowest average coverage depth and the SNV detection limit from LRS to page 11-12, line 393-403. 

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the author evaluates the use of PacBio long-read sequencing for detecting mitochondrial DNA (mtDNA) variants, including SNVs and large-scale deletions, and compares its performance with conventional short-read sequencing approaches. The study shows that long-read sequencing can accurately detect mtDNA deletions, determine breakpoints, and estimate heteroplasmy levels. The work highlights the potential advantages of long-read sequencing for resolving complex mtDNA structural variants.

After reading through the manuscript, My major comments are:

1. Some figures in the manuscript are difficult to read because the font size of labels and annotations is very small. It would be helpful if the authors could enlarge the text in the figures to improve readability.
2. It would be useful if the authors could discuss in more detail how sequencing depth influences the detection of mtDNA mutations and large deletions in short-read versus long-read sequencing. Short-read sequencing can usually achieve much higher depth at the same sequencing cost. Since heteroplasmy detection is highly dependent on depth, a discussion of how sequencing depth affects the sensitivity of these two approaches would strengthen the manuscript.
3. In this study, sequencing was performed on total genomic DNA without specific enrichment of mtDNA. The authors may consider discussing whether isolating or enriching mtDNA prior to sequencing could improve the detection of mtDNA mutations or deletions. In particular, it would be interesting to compare long-read sequencing of total DNA with short-read sequencing performed on mtDNA-enriched samples, if such comparisons have been reported.
4. It would be helpful if the authors could include more intuitive examples showing the read coverage patterns for both sequencing methods around mtDNA mutation sites or deletion breakpoints (for example, IGV views). This would allow readers to more clearly visualize the differences between short-read and long-read sequencing in detecting mtDNA variants.

 

 

Author Response

Comments 1: Some figures in the manuscript are difficult to read because the font size of labels and annotations is very small. It would be helpful if the authors could enlarge the text in the figures to improve readability.

Response 1: Thank you for this suggestion. We have updated all figures with larger font sizes. 

Comments 2: It would be useful if the authors could discuss in more detail how sequencing depth influences the detection of mtDNA mutations and large deletions in short-read versus long-read sequencing. Short-read sequencing can usually achieve much higher depth at the same sequencing cost. Since heteroplasmy detection is highly dependent on depth, a discussion of how sequencing depth affects the sensitivity of these two approaches would strengthen the manuscript.

Response 2: We appreciate the reviewer’s suggestion. We have added a more detailed descriptive sensitivity analysis based on our downsampling experiment to the manuscript results page 7 line 217-226. 

Comments 3: In this study, sequencing was performed on total genomic DNA without specific enrichment of mtDNA. The authors may consider discussing whether isolating or enriching mtDNA prior to sequencing could improve the detection of mtDNA mutations or deletions. In particular, it would be interesting to compare long-read sequencing of total DNA with short-read sequencing performed on mtDNA-enriched samples, if such comparisons have been reported.

Response 3: We appreciate the reviewer’s valuable suggestion. Isolating mitochondria or enriching mtDNA can indeed enhance sequencing depth and increase detection sensitivity. However, mitochondrial isolation demands substantial tissue and incurs high costs, making it impractical for clinical applications and not feasible for commonly used tissue types such as blood, saliva and urine. On the other hand, mtDNA enrichment can be achieved using PCR methods, similar to what we employed for SRS. A recent publication by Lin et al. utilized LR-PCR for mtDNA enrichment, followed by Illumina SRS and PacBio LRS (PMID: 19546809). However, this approach inherits the amplification bias from LR-PCR, rendering it incapable of accurately quantifying mtDNA deletion heteroplasmy. We have referenced this study and discussed its limitations and advantages in the discussion (page 11-12, line 393--403). 

Comments 4: It would be helpful if the authors could include more intuitive examples showing the read coverage patterns for both sequencing methods around mtDNA mutation sites or deletion breakpoints (for example, IGV views). This would allow readers to more clearly visualize the differences between short-read and long-read sequencing in detecting mtDNA variants.

Response 4: Thanks for the suggestion. We’ve created coverage plots from SRS and LRS for all samples and added them to Supplementary Figure 5. We’ve grouped them into three categories: A. coverage for SLSMD samples, B. coverage for MMD samples, and C. coverage for deletion-negative control samples. 

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript by Jadhav et al aims to compare the PacBio Long Read Sequencing (LRS) technique with the most frequently used method of long-range PCR-based targeted mtDNA sequencing by Short Read Sequencing (SRS) in terms of accurate genetic diagnosis of primary mitochondrial diseases. While quantifying the heteroplasmic load of mtDNA pathogenic variants is critically important for patients with a suspected primary inherited mitochondrial disease, the scientific value of their study is significantly diminished being a replication study of the 2024 published study by Lin et al. The authors failed to justify why they replicated the Lin et al., 2024 study. In fact, the authors superficially discussed their findings in the context of the original study by citing it late in the discussion section.

Would the authors have investigated the power of the PacBio Long Read Sequencing (LRS) technique in the context of single nucleotide variants (SNVs) with very low heteroplasmic load (less than 10%), it could have justified their replicate study in terms of clinical applicability for patients with a suspected primary inherited mitochondrial disease.  This would improve the diagnosis rate for these patients, especially in the context of “genetic first-based approach” to unravel novel pathogenetic mechanisms of primary mitochondrial diseases caused by pathogenic mtDNA variants, the main objective articulated by the authors in the introduction section.

Author Response

Comments 1: 

The manuscript by Jadhav et al aims to compare the PacBio Long Read Sequencing (LRS) technique with the most frequently used method of long-range PCR-based targeted mtDNA sequencing by Short Read Sequencing (SRS) in terms of accurate genetic diagnosis of primary mitochondrial diseases. While quantifying the heteroplasmic load of mtDNA pathogenic variants is critically important for patients with a suspected primary inherited mitochondrial disease, the scientific value of their study is significantly diminished being a replication study of the 2024 published study by Lin et al. The authors failed to justify why they replicated the Lin et al., 2024 study. In fact, the authors superficially discussed their findings in the context of the original study by citing it late in the discussion section.

Would the authors have investigated the power of the PacBio Long Read Sequencing (LRS) technique in the context of single nucleotide variants (SNVs) with very low heteroplasmic load (less than 10%), it could have justified their replicate study in terms of clinical applicability for patients with a suspected primary inherited mitochondrial disease.  This would improve the diagnosis rate for these patients, especially in the context of “genetic first-based approach” to unravel novel pathogenetic mechanisms of primary mitochondrial diseases caused by pathogenic mtDNA variants, the main objective articulated by the authors in the introduction section.

Response 1: We sincerely thank the reviewer for their thoughtful comments and for highlighting the comparison with the 2024 study by Lin et al. We appreciate this opportunity to clarify the rationale and distinct contributions of our work. While both studies compare mtDNA sequencing results generated by Illumina short-read sequencing (SRS) and PacBio long-read sequencing (LRS), there are several important methodological and analytical differences that distinguish our study and provide additional value. 

1. Differences in experimental design and bias in heteroplasmy quantification: In the study by Lin et al., long-range PCR (LR-PCR) products were used for both SRS and LRS. As a result, both approaches inherit amplification bias from LR-PCR, which limits accurate quantification of mtDNA deletion heteroplasmy. In contrast, our study utilized total genomic DNA and performed PacBio LRS without PCR amplification. This amplification-free approach minimizes bias and enables more accurate quantification of large mtDNA deletion heteroplasmy. 
2. Clarification of deletion heteroplasmy discrepancies:Lin et al. did not clearly address discrepancies between SRS and LRS in deletion heteroplasmy measurements. In particular, the methodology used to derive deletion heteroplasmy values in their Table 2 is not fully transparent. Although real-time PCR was described for structural variant (SV) quantification, the primer design (targeting ND1 and ND4) appears suitable only for one specific case, the single-fiber analysis in sample P7, and may not be applicable to many deletion samples where both regions are encompassed within the deletion. In contrast, our study systematically evaluated heteroplasmy using genome-based LRS alongside quantitative PCR, explicitly addressing known limitations of qPCR, which has historically been considered a gold standard. 
3. Distinction between single and multiple mtDNA deletions:The Lin et al. study categorized deletions broadly as SVs without distinguishing between single and multiple mtDNA deletions. However, this distinction is clinically and biologically important, as single deletions are typically associated with primary mtDNA deletion syndromes, whereas multiple deletions are often secondary to nuclear gene defects, degenerative processes, or aging. Several samples in their dataset (e.g., those with clinical features of IIM and MM) may represent multiple deletions, where reported heteroplasmy reflects only a single deletion species rather than the overall deletion burden. In contrast, our study developed a dedicated bioinformatics pipeline capable of detecting and differentiating single versus multiple deletions. Additionally, our LRS approach enables precise breakpoint mapping, confirming that the common 4977 bp deletion is the most prevalent deletion observed in mitochondrial myopathies. 
4. SNV detection sensitivity and low-level heteroplasmy: Both studies achieved comparable sensitivity for SNV detection (~5%), despite differences in variant-calling tools. While LoFreq did not detect one variant at 6% heteroplasmy (m.5791G>A), it successfully identified other low-level variants, including m.13897T>C at 4.7% (M7) and m.16327C>T at 6.1% (M6), as now clarified in the revised manuscript (page 7, lines 195–200). We agree with the reviewer that evaluating very low-level heteroplasmy (<10%) is clinically important, and our results support the utility of LRS in this context.  

In summary, although our study shares a general comparative framework with Lin et al., it differs in experimental design, analytical methodology, and clinical interpretation. Specifically, our amplification-free LRS approach, improved heteroplasmy quantification, and the ability to distinguish and characterize deletion types provide additional insights relevant to clinical diagnostics. We have now expanded the discussion to more explicitly contextualize our findings relative to Lin et al. (page 11, lines 361–369). 

We appreciate the reviewer’s constructive feedback, which has helped us strengthen the manuscript. 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewer concerns have been fully addressed, the manuscript can be accepted for publication now. 

Author Response

Comments 1: The reviewer concerns have been fully addressed, the manuscript can be accepted for publication now. 

Response 1: Thank you for your thorough review and for confirming that our manuscript has addressed all your concerns. We greatly appreciate your time and constructive feedback throughout the revision process. Your insights were invaluable in improving the clarity and quality of our work.  

Reviewer 4 Report

Comments and Suggestions for Authors

Although the authors’ revisions clarified key points from the original submission, the following weaknesses still need to be addressed:

• The title of the manuscript is misleading with the inclusive term of mtDNA variants, when in fact the revised version focuses on the main advantage of PacBio Long-Read sequencing dedicated for single large-scale mtDNA deletions and multiple mtDNA deletions.
• The revised abstract has better scope, but the high density of undefined abbreviations makes it hard to follow. Please define all abbreviations, ensure consistency, and clarify 'advanced mtDNA analysis' in the final sentence.
• The introduction requires revision to incorporate the feedback and address the limitations highlighted in the initial critique. The introduction still misses a compelling rationale for the new method, and why the technology developed by Lin et al is not adequate.
• The first sentence of the discussion is awkward, as a study itself cannot perform an analysis.
• Dedicate the first paragraph of the discussion to the 'so what': Why is this new method necessary? Focus on challenging the current gold standard by highlighting its weaknesses and outlining how this novel approach fills a critical void in mtDNA genetic analysis in a clinical context.

Author Response

Although the authors’ revisions clarified key points from the original submission, the following weaknesses still need to be addressed:

Comments 1: The title of the manuscript is misleading with the inclusive term of mtDNA variants, when in fact the revised version focuses on the main advantage of PacBio Long-Read sequencing dedicated for single large-scale mtDNA deletions and multiple mtDNA deletions.

Response 1: While this study primarily focuses on detecting large-scale mtDNA deletions, we also conducted a thorough evaluation of SNV detections in our PacBio LRS data. We have included detailed results for these SNVs in the manuscript and supplementary materials. In response to the reviewer’s suggestion, we have revised the title to: “Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA Variant Detection, with a specific emphasis on mtDNA deletion detection and quantification”. 

Comments 2: The revised abstract has better scope, but the high density of undefined abbreviations makes it hard to follow. Please define all abbreviations, ensure consistency, and clarify 'advanced mtDNA analysis' in the final sentence.

Response 2: All abbreviations have been defined at their first occurrence in the abstract. Furthermore, we have changed “advanced” in the last sentence to “comprehensive”. Our results demonstrated that PacBio LRS can accurately detect mtDNA sequencing variants and deletions, as well as provide heteroplasmy information. 

Comments 3: The introduction requires revision to incorporate the feedback and address the limitations highlighted in the initial critique. The introduction still misses a compelling rationale for the new method, and why the technology developed by Lin et al is not adequate.

Response 3: We appreciate the reviewer’s suggestion. We’ve incorporated rationale of PacBio LRS and addressed the differences between Lin’s study and our study in introduction (Page 2, Lines 77-92). 

Comments 4: The first sentence of the discussion is awkward, as a study itself cannot perform an analysis. Dedicate the first paragraph of the discussion to the 'so what': Why is this new method necessary? Focus on challenging the current gold standard by highlighting its weaknesses and outlining how this novel approach fills a critical void in mtDNA genetic analysis in a clinical context.

Response 4: Thank you for your thoughtful feedback. We have removed the original first sentence of the discussion. Additionally, we revised the first two paragraphs (pages 9–10, lines 273–302) to emphasize the significance of our findings. The updated discussion now opens by answering the 'so what' question, clearly explaining why our new method is necessary. It directly addresses the limitations of the current gold standard for mtDNA genetic analysis in clinical settings and highlights how our approach fills this critical gap. 

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed the majority of concerns, thereby improving their revised manuscript. However, the following comments need to be adequately addressed:

1) Comment #1: The authors responded positively to the suggestion of amending their title "we have revised the title to: “Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA Variant Detection, with a specific emphasis on mtDNA deletion detection and quantification”. However, the revised title reads as: "Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA Variant Detection, focusing on mtDNA deletion detection and quantification". The revised title articulated in their rebuttal letter is better than the one in the revised version, with the caveat of defining the abbreviation of mtDNA. The revised title should read "Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA (mtDNA) Variant Detection, with an Emphasis on Detection and Quantification of mtDNA Deletion"

Comment #2: All except one abbreviations have been defined in the revised abstract. Please define "ddPCR". In addition, the last sentence should be complemented by the argument encapsulated in their response #2:" In conclusion, our findings establish PacBio LRS as a robust tool for comprehensive mtDNA analysis capable of accurately detecting and quantifying heteroplasmic mtDNA variants and complex deletions".

Comment #3: The authors adequately addressed the concern encapsulated in the previous comments to the authors regarding their rationale of their study vis-à-vis the Lin et al study.

Comment #4: The authors greatly improved the first two paragraphs of the discussion section. Thus, the revised discussion is more forceful, lending credence for the need to consider this new method for analyzing mtDNA variants and deletions in a clinical setting.

 

Author Response

The authors have addressed the majority of concerns, thereby improving their revised manuscript. However, the following comments need to be adequately addressed:

Comment #1: The authors responded positively to the suggestion of amending their title "we have revised the title to: “Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA Variant Detection, with a specific emphasis on mtDNA deletion detection and quantification”. However, the revised title reads as: "Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA Variant Detection, focusing on mtDNA deletion detection and quantification". The revised title articulated in their rebuttal letter is better than the one in the revised version, with the caveat of defining the abbreviation of mtDNA. The revised title should read "Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA (mtDNA) Variant Detection, with an Emphasis on Detection and Quantification of mtDNA Deletion"

Response #1: We have updated the manuscript title to the revised version suggested by the reviewer. 

Comment #2: All except one abbreviations have been defined in the revised abstract. Please define "ddPCR". In addition, the last sentence should be complemented by the argument encapsulated in their response #2:" In conclusion, our findings establish PacBio LRS as a robust tool for comprehensive mtDNA analysis capable of accurately detecting and quantifying heteroplasmic mtDNA variants and complex deletions".

Response #3: We have updated the abstract to define ddPCR as “Droplet Digital PCR (ddPCR)”. The last sentence of the abstract has been updated to the sentence suggested by the reviewer.  

Comment #3: The authors adequately addressed the concern encapsulated in the previous comments to the authors regarding their rationale of their study vis-à-vis the Lin et al study.

Comment #4: The authors greatly improved the first two paragraphs of the discussion section. Thus, the revised discussion is more forceful, lending credence for the need to consider this new method for analyzing mtDNA variants and deletions in a clinical setting.

Response #3 and #4: Thank you for your thorough review and for confirming that our manuscript has addressed all your concerns. We greatly appreciate your time and constructive feedback throughout the revision process. Your insights were invaluable in improving the clarity and quality of our work.