Advancing Drug Resistance Detection: Comparative Analysis Using Short-Read and Long-Read Next-Generation Sequencing Technologies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study titled "Advancing Drug Resistance Detection: Comparative Analysis Using Short-read and Long-read Next-generation Sequencing Technologies" compares the results of sequencing viral nucleic acid sequences using different sequencing methods, contributing to the development of viral diagnostic technologies. However, the descriptions of experimental methods and results are overly generalized and lack standardization, raising concerns about the rigor of the methods, the standardization of the results, and the reliability of the conclusions.

Major issues

• The description of the "Extraction of viral DNA/RNA" methodology section is overly generalized, which affects the reproducibility of this experimental approach. It is recommended to provide detailed experimental procedures and parameters to assist readers in understanding how viral DNA or RNA was extracted in this study.
• The section "Amplification of target regions by RT-PCR and PCR" should provide the sequence information of the used primers.
• Considering that this study primarily focuses on comparing different sequencing methods, the descriptions of "2.4. Illumina sequencing," "2.5. Nanopore sequencing," and "2.6. MGI sequencing" are overly generalized, with almost no mention of the key experimental procedures and critical experimental parameters in sequencing.
• The software involved in the experimental methods should provide the version number.
• Table 2 and Table 3 lack column names, thereby reducing the readability of the tables. Similarly, the readability of Table 5, 6, and 7 is also relatively low.
• Considering the biological and technical replicates, the data presented in Table 4 are nonstandard. It is recommended to present them in the form of "mean ± error".
• Figure 2 appears blurred. Additionally, the figure legends for Figure 2 lack clear annotations. For example, symbols such as "N0" and "1" in the figure should be annotated. Similar issues exist in Figures 3, 4, and 5.
• "The description of the results section in '3.2. Global comparative analysis between different platforms' is not clear enough and causes confusion. Since different sequencing methods have determined the nucleic acid sequences of several distinct viruses, there should be a specific quantitative description of the results obtained from applying different sequencing methods to sequence the nucleic acid sequences of different viruses."
• According to the principle of data availability, the raw sequencing data should be uploaded to public data platforms.

Minor issues

• The first occurrence of an abbreviation should be accompanied by its full name, such as "ABL" on the first page, and "NGS" and "MGI" on the second page.
• The closing parenthesis ")" in "...for drug resistance)" on the first page is unnecessary.

Author Response

We are grateful to the reviewer for their constructive and thoughtful feedback.

The English has been corrected.

Major issues

Comments 1: The description of the "Extraction of viral DNA/RNA" methodology section is overly generalized, which affects the reproducibility of this experimental approach. It is recommended to provide detailed experimental procedures and parameters to assist readers in understanding how viral DNA or RNA was extracted in this study.

Response 1: Information was added in the "Extraction of viral DNA/RNA" part in methodology section.

Comments 2: The section "Amplification of target regions by RT-PCR and PCR" should provide the sequence information of the used primers.

Response 2: The primer sequences used in this study are proprietary. As such, we are currently unable to disclose the full sequences publicly. However, we confirm that all primers were designed to target highly conserved regions, and their performance has been validated experimentally in terms of sensitivity and specificity.

Comments 3: Considering that this study primarily focuses on comparing different sequencing methods, the descriptions of "2.4. Illumina sequencing," "2.5. Nanopore sequencing," and "2.6. MGI sequencing" are overly generalized, with almost no mention of the key experimental procedures and critical experimental parameters in sequencing.

Response 3: Additional, more precise information on the various sequencing protocols has been added to the 3 sections mentioned.

Comments 4: The software involved in the experimental methods should provide the version number.

Response 4: DeepChek®-HCV v2.0 was added to the material and method part.

Comments 5: Table 2 and Table 3 lack column names, thereby reducing the readability of the tables. Similarly, the readability of Table 5, 6, and 7 is also relatively low.

Response 5: In the case of Table 2, this is deliberate, as all the columns after the first relate to the size of the amplified base-pair regions. For Table 3, the column headings have been added. Layout modifications have been made to Tables 5, 6 and 7 to make them more readable.

Comments 6: Considering the biological and technical replicates, the data presented in Table 4 are nonstandard. It is recommended to present them in the form of "mean ± error".

Response 6: Thank you for your suggestion. We would like to clarify that the data presented in Table 4 correspond to the performance metrics obtained from a single representative run on each sequencing platform. These values are not derived from biological or technical replicates but rather aim to provide a descriptive comparison of the output quality between instruments. For this reason, the values are presented as absolute numbers and not as "mean ± error". We have now clarified this point in the manuscript to avoid any misunderstanding.

Comments 7: Figure 2 appears blurred.

Response 7: Figure 2 has been removed from the revised version of the manuscript. To address the point it illustrated, we have added the following sentence to the methods section:

"Libraries were quality-checked using TapeStation, and the fragment size distribution ranged from 150 to 900 bp."

Comments 8: Additionally, the figure legends for Figure 2 lack clear annotations. For example, symbols such as "N0" and "1" in the figure should be annotated. Similar issues exist in Figures 3, 4, and 5.

Response 8: Figure 2 has been removed from the revised version of the manuscript.

Comments 9: "The description of the results section in '3.2. Global comparative analysis between different platforms' is not clear enough and causes confusion. Since different sequencing methods have determined the nucleic acid sequences of several distinct viruses, there should be a specific quantitative description of the results obtained from applying different sequencing methods to sequence the nucleic acid sequences of different viruses."

Response 9: Thank you for your valuable feedback. We would like to clarify that the objective of section 3.2 was not to evaluate the sequencing performance of each platform virus by virus, but rather to compare the ability of the different platforms to detect both major and minor variants across all applications. By pooling the variant detection data from the various viruses, we aimed to assess the global detection of each technology. This approach allowed us to strengthen the statistical power of our comparisons and to highlight significant differences between platforms in terms of variant detection capabilities

Comments 10: According to the principle of data availability, the raw sequencing data should be uploaded to public data platforms.

Response 10: Thank you for your comment. We acknowledge the principle of data availability. However, the raw sequencing data in this study were generated using external quality assessment material from QCMD. According to QCMD’s terms of use and confidentiality policy, the raw data derived from these panels cannot be shared publicly. These materials are intended for performance evaluation and benchmarking only, and public deposition is restricted.

 

Minor issues

Comments 11: The first occurrence of an abbreviation should be accompanied by its full name, such as "ABL" on the first page, and "NGS" and "MGI" on the second page.

Response 11: The correction has been made

Comments 12: The closing parenthesis ")" in "...for drug resistance)" on the first page is unnecessary.

Response 12: The parenthesis has been removed

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents valuable findings but requires further refinement to enhance its clarity and overall impact. Additional improvements in structure or detail would strengthen the study.

Major Comments: 

The authors are advised to rewrite the entire methodology section more clearly, specifying the purpose / principle of each step. This will help readers understand the full process and make it easier to reproduce the results if they wish to do so. In section 2.8 Statistical tests: What is the Tuckey test? I believe it should be spelled Tukey test. Also, the purpose of both the Tukey and Sidak tests is not clearly explained. The authors should rewrite this entire paragraph to make it clearer. Figure 2 is unclear, kindly recreate or improve the figure for better clarity.

Minor Comments: 

In this manuscript authors should improve the flow of the text. Please correct typographical errors. For instance, in the abstract, "Sars-Cov-2" should be written as "SARS-CoV-2 like the author has correctly written in the first line of Background. Another example is the phrase “andMk1C MinION” in the abstract, there should be a space after “and".  Authors are advised to carefully review and correct such minor mistakes.” The page numbers in the article are incorrect. For example, "2 of 20" repeated multiple times in different pages.

Author Response

We are grateful to the reviewer for their constructive and thoughtful feedback.The English has been corrected.

 

Major Comments:

Comments 1: The authors are advised to rewrite the entire methodology section more clearly, specifying the purpose / principle of each step. This will help readers understand the full process and make it easier to reproduce the results if they wish to do so.

Response 1: All of the methodology part was review with more explanations.

Comments 2: In section 2.8 Statistical tests: What is the Tuckey test? I believe it should be spelled Tukey test. Also, the purpose of both the Tukey and Sidak tests is not clearly explained. The authors should rewrite this entire paragraph to make it clearer.

Response 2: The whole paragraph has been rewritten.

Comments 3: Figure 2 is unclear, kindly recreate or improve the figure for better clarity.

Response 3: Figure 2 has been removed from the revised version of the manuscript. To address the point it illustrated, we have added the following sentence to the methods section:

"Libraries were quality-checked using TapeStation, and the fragment size distribution ranged from 150 to 900 bp."

 

Minor Comments:

Comments 4: In this manuscript authors should improve the flow of the text. Please correct typographical errors. For instance, in the abstract, "Sars-Cov-2" should be written as "SARS-CoV-2 like the author has correctly written in the first line of Background.

Response 4: SARS-CoV-2 has been corrected in the first line of Background

Comments 5: Another example is the phrase “andMk1C MinION” in the abstract, there should be a space after “and". 

Response 5: “andMk1C MinION” has been corrected

Comments 6: Authors are advised to carefully review and correct such minor mistakes.” The page numbers in the article are incorrect. For example, "2 of 20" repeated multiple times in different pages.

Response 6: the number of page has been corrected

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Advancing Drug Resistance Detection: Comparative Analysis Using Short-read and Long-read Next-generation Sequencing Technologies.” covers an important topic, however, this study is not supported by ethics approval or institutional review board approval. As patients (page 2; Tables 5-6) are mentioned throughout this paper, ethics approval from an ethics committee or institutional review board needs to be clearly stated in this manuscript.  If patient-level data was deidentified that should be clearly mentioned within the informed consent statement,  confirming consent was not applicable. At least three different samples for each microorganism (including the control) should have been anlayzed to confirm reproducibility/robustness of the results obtained.  Figure 2 (Page 9) is blurry and these images and  X/Y axes cannot be read. In text citations are not cited in MDPI format and they should be within square brackets [ ].  Superscripted page numbers on top right hand side of pages are incorrect for many pages. Page 5 has no page listed and page 6 and onwards goes back to page 2 or other numbers that have already been used.

Minor comments:

Title should not end with a full stop/period

Abstract: mycobacterium tuberculosis should be Mycobacterium tuberculosis

Listed keywords shouldn’t be abbreviations. Consider adding an abbreviations list to this manuscript.

Page 2:  “And, on Nanopore, sequencing ……..”

sentences should not start with “And”

In Table 1, I recommend using scientific notation for viral load (10³  instead of 10e3) and decimal places instead of commas for entries 13-15. i.e. 3,89*10e3 should be 3.89 X 10³.

Table 4. should commas be used for the numbers listed? Or should this be a decimal point .

Start of section 4.2—1 sentence is not a paragraph

Comments on the Quality of English Language

There are numerous grammatical inconsistencies/errors throughout the manuscript. 

Author Response

We are grateful to the reviewer for their constructive and thoughtful feedback.The English has been corrected.

 

Comments 1: The manuscript entitled “Advancing Drug Resistance Detection: Comparative Analysis Using Short-read and Long-read Next-generation Sequencing Technologies.” covers an important topic, however, this study is not supported by ethics approval or institutional review board approval. As patients (page 2; Tables 5-6) are mentioned throughout this paper, ethics approval from an ethics committee or institutional review board needs to be clearly stated in this manuscript.  If patient-level data was deidentified that should be clearly mentioned within the informed consent statement, confirming consent was not applicable.

Response 1: We apologize for the misstatement. In fact, the data mentioned in the study refer to QCMD (Quality Control for Molecular Diagnostics) external quality controls, not clinical samples from human subjects. These are standardized, testing materials provided for performance evaluation purposes only. Therefore, Institutional Review Board (IRB) approval was not required, as no human subjects were involved.

Comments 2: At least three different samples for each microorganism (including the control) should have been anlayzed to confirm reproducibility/robustness of the results obtained.

Response 2: Thank you for this important observation. We acknowledge that analyzing multiple independent samples per microorganism would strengthen the assessment of reproducibility and robustness. However, in this study, the primary objective was to perform a comparative evaluation of sequencing platforms using standardized external quality assessment (EQA) materials from QCMD. These panels are designed to provide a single representative sample per target organism, under controlled conditions. Although the number of samples per microorganism was limited, each was processed in parallel across all platforms, allowing for consistent comparative analysis.

Comments 3: Figure 2 (Page 9) is blurry and these images and  X/Y axes cannot be read.

Response 3: Figure 2 has been removed from the revised version of the manuscript. To address the point it illustrated, we have added the following sentence to the methods section:

"Libraries were quality-checked using TapeStation, and the fragment size distribution ranged from 150 to 900 bp."

Comments 4: In text citations are not cited in MDPI format and they should be within square brackets [ ]. 

Response 4: Citation format has been corrected

Comments 5: Superscripted page numbers on top right hand side of pages are incorrect for many pages. Page 5 has no page listed and page 6 and onwards goes back to page 2 or other numbers that have already been used.

Response 5: Page number has been corrected

 

Minor comments:

Comments 6: Title should not end with a full stop/period

Response 6: The period has been removed.

Comments 7: Abstract: mycobacterium tuberculosis should be Mycobacterium tuberculosis

Response 7: It has been corrected

Comments 8: Listed keywords shouldn’t be abbreviations. Consider adding an abbreviations list to this manuscript.

Response 8: Keywords list has been added

Comments 9: Page 2:  “And, on Nanopore, sequencing ……..”

sentences should not start with “And”

Response 9: The sentence has been changed.

Comments 10: In Table 1, I recommend using scientific notation for viral load (103  instead of 10e3) and decimal places instead of commas for entries 13-15. i.e. 3,89*10e3 should be 3.89 X 103.

Response 10: Scientific notation has been applied.

Comments 11: Table 4. should commas be used for the numbers listed? Or should this be a decimal point .

Response 11: We have modified and added a decimal point.

Comments 12: Start of section 4.2—1 sentence is not a paragraph

Response 12: Thank you for your observation. We have expanded the first sentence of section 4.2 to form a complete paragraph, providing additional context on the importance of molecular biology techniques in studying infectious diseases.

 

“The study of HIV-1, HBV, HCV, SARS-CoV-2, and TB relies predominantly on molecular biology techniques, which typically include viral or bacterial genome amplification and sequencing. These approaches are essential for accurate detection, genotyping, and the identification of resistance-associated mutations. Advances in sequencing technologies have significantly improved the sensitivity and throughput of such analyses, making them indispensable tools for both clinical diagnostics and epidemiological surveillance.”

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I express my gratitude to the authors for incorporating the suggested revisions, which have enhanced the manuscript's quality. However, it is unfortunate that the authors have not provided the primer sequences used in the sequencing process. This omission hinders the ability to assess the experimental design's rationale and the sequencing results' reliability. Additionally, the inability to upload the sequencing data to a third-party database for public access compromises the verifiability of the research findings and fails to comply with established data-sharing policies.

Author Response

Comments : I express my gratitude to the authors for incorporating the suggested revisions, which have enhanced the manuscript's quality. However, it is unfortunate that the authors have not provided the primer sequences used in the sequencing process. This omission hinders the ability to assess the experimental design's rationale and the sequencing results' reliability. Additionally, the inability to upload the sequencing data to a third-party database for public access compromises the verifiability of the research findings and fails to comply with established data-sharing policies.

 

Response: We sincerely thank the reviewer for their continued engagement and constructive feedback, which have clearly contributed to enhancing the overall quality and clarity of our manuscript.

1/ Primer Sequences

Regarding the primer sequences, we respectfully inform the reviewer that these are part of proprietary commercial kits developed and distributed by certified diagnostic manufacturers. These kits are protected by intellectual property rights, and their detailed composition, including primer sequences, is not publicly disclosed by the manufacturers. We are contractually and ethically bound by these confidentiality clauses, and therefore not authorized to share such sequences.

However, we fully recognize the importance of reproducibility and scientific transparency. To address this, we have included in the revised version of the manuscript a detailed list of all commercial kits used, complete with product names, catalogue numbers, and suppliers. This enables other researchers to replicate the experimental procedures under equivalent conditions by sourcing the exact same validated kits.

Moreover, it is worth noting that the use of CE-IVD or RUO commercial kits is standard practice in many applied studies and is commonly accepted in the literature, especially when dealing with validated tools whose performance has been independently evaluated.

2/QCMD Raw Data

Concerning the raw data from the QCMD panels, we fully understand the value of open data sharing. However, the QCMD program is part of a regulated external quality assessment (EQA) framework, in which participating laboratories receive standardized samples under strict confidentiality agreements. The raw data generated from these panels are intended for internal proficiency evaluation only and remain under the ownership and control of QCMD.

As such, we are not legally permitted to upload or disclose raw sequencing data from QCMD samples in public databases. This restriction is clearly stated in QCMD’s participation policy and applies uniformly to all users.

To support the validity of our work despite this limitation, we have sendes the official QCMD certificates. Furthermore, we have indicated that interested researchers can contact QCMD directly to request access to archived panels or to participate in future evaluations using equivalent materials.

We hope that these additional explanations help clarify our constraints and demonstrate that we have taken all reasonable steps to ensure methodological transparency and reproducibility, within the legal and ethical boundaries imposed by the tools and data providers involved.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for submitting the revised manuscript.

Author Response

We sincerely thank the reviewer for their continued engagement

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Advancing Drug Resistance Detection: Comparative Analysis Using Short-read and Long-read Next-generation Sequencing Technologies” covers an important topic and concerns pertaining to samples has been clarified. The following comments need to get addressed:

In abstract  Mycobacterium Tuberculosis should be Mycobacterium tuberculosis  and be italicized.

Page 1.  Corresponding author symbol is not placed next to corresponding author.

Section 2.1. The yellow highlighted section: “…. In 13 patients and was unavailable in the remaining 2 patients.” has not been reworded in comparison to draft 1.  I strongly recommend for the patient wording to be modified as clinical samples were not analyzed.

The page number inconsistency in top right corner of pages has not been resolved. Why is page 6 still listed as page 2 and page 7 is listed as page 3? There are other inconsistencies pertaining to page numbers.

Section 2.4 (page 6). Add a space after (ABL Diagnostics)

 

Table 1. Scientific notation has not be utilized X103 should be X 10³ and X 101 and X 108 should be X 10¹ and X 10⁸, respectively. 

Table 3. Mycobacterium Tuberculosis should be Mycobacterium tuberculosis and be italicized.

Section 3.3. “Table 5.Samples” Should be “Table 5. Samples”   (add a space after period).

Tables  5 & 6. Title. Consider rewording patient terminology.

Table 5 is now distorted. Pages 15 and 16 have no data and pages 17-18 is confusing as currently presented. Font size for column titles should be the same.

Table 6 is also distorted and column subtitles /data need to get reformatted.

Pages 24 and 26. Mycobacterium Tuberculosis should be Mycobacterium tuberculosis and be italicized.

Page 27. Conflicts of Interest statement needs to be separated from Abbreviations list.

Author Response

We sincerely thank you for your second review of the manuscript and for your insightful comments, which have contributed to enhancing the quality of the work.

Comments 1: : In abstract  Mycobacterium Tuberculosis should be Mycobacterium tuberculosis  and be italicized.

Response 1: Italic notation has been applied to all parts containing Mycobacterium tuberculosis.  

Comments 2: Page 1.  Corresponding author symbol is not placed next to corresponding author.

Response 2: It has been corrected.

Comments 3: Section 2.1. The y ellow highlighted section: “…. In 13 patients and was unavailable in the remaining 2 patients.” has not been reworded in comparison to draft 1.  I strongly recommend for the patient wording to be modified as clinical samples were not analyzed.

Response 3: The word “patient” has been replaced by “QCMD” for greater consistency and precision.

Comments 4: The page number inconsistency in top right corner of pages has not been resolved. Why is page 6 still listed as page 2 and page 7 is listed as page 3? There are other inconsistencies pertaining to page numbers.

Response 4: We would like to clarify that this issue appears to be related to the journal’s formatting template or header settings, rather than the manuscript file itself. Despite our attempts, we have not been able to resolve the pagination problem on our end.

We therefore kindly suggest that the journal's production team guide us on the appropriate steps or implement the necessary corrections during the typesetting stage.

Comments 5: Section 2.4 (page 6). Add a space after (ABL Diagnostics)

Response 5: It has been corrected.

Comments 6: Table 1. Scientific notation has not be utilized X103 should be X 10³ and X 101 and X 108 should be X 10¹ and X 10⁸, respectively. 

Response 6: It has been corrected.

Comments 7: Table 3. Mycobacterium Tuberculosis should be Mycobacterium tuberculosis and be italicized.

Response 7: It has been corrected.

Comments 8: Section 3.3. “Table 5.Samples” Should be “Table 5. Samples”   (add a space after period).

Response 8: It has been corrected.

Comments 9: Tables  5 & 6. Title. Consider rewording patient terminology.

Response 9: It has been corrected.

Comments 10:Table 5 is now distorted. Pages 15 and 16 have no data and pages 17-18 is confusing as currently presented. Font size for column titles should be the same.

Response 10: It seems that the format is modified according to the version of Word used. The table format has been revised.

Comments 11: Table 6 is also distorted and column subtitles /data need to get reformatted.

Response 11: The table format has been revised.

Comments 12: Pages 24 and 26. Mycobacterium Tuberculosis should be Mycobacterium tuberculosis and be italicized.

Response 12: It has been corrected..

Comments 13: Page 27. Conflicts of Interest statement needs to be separated from Abbreviations list. Response 13: It has been corrected.