Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Metagenomic and Targeted Next-Generation Sequencing Technologies in Infectious Disease Diagnostics: Current Applications, Challenges, and Future Perspectives

 

Suggest changing the title as follows

“Metagenomic and targeted next-generation sequencing in infectious disease diagnostics: current applications, challenges, and future perspectives”

 

Introduction

1. In line 48-50, the authors mention that microbiological culture requires 24 hours for pathogen growth but have not mentioned the time required for mNGS. Suggest mentioning the turnaround time for mNGS also explicitly highlighting its rapidity compared to culture method.
2. In addition to mNGS, introducing targeted NGS also would be beneficial for the authors.
3. In line number 84 to 92, technical challenges are mentioned. In addition to that, I suggest adding about the handling of commensal organisms also.

 

Next-Generation Sequencing Technologies

1. In general, the authors have provided a good technical breakdown of sequencing methods.
2. In section 2.2, suggest mentioning about the limitation of detection of tNGS in comparison with the mNGS and the clinical reasons for choosing these two techniques at specific instances.
3. In section 2.1.1, line 128-130, the authors should briefly acknowledge the limitations of RNA sequencing. Eg; complexity, cost, cold chain requirement etc. also
4. In section 2.1.2, it would be beneficial to mention how accuracy and error rates vary among SGS and TGS.
5. In section 2.2.1.2, mentioning these data in words may affect readability. It is better to use a table to highlight the importance of mNGS, hc-tNGS, and mp-tNGS techniques.
6. Scientific names of organisms should be italicized throughout the manuscript. Eg. Streptococcus pneumoniae in line 258, 760, 764, 767 etc.
7. In line 261-264, authors have mentioned regarding mNGS performance among a group of patients who received empiric antibiotic treatment. Considering the significance of this point, I suggest emphasizing more on the importance mNGS testing in patients who have exposed to antibiotics prior to hospital admission and showing culture-negative results.
8. In line 284-286, authors state that detecting of low viral load in viral encephalitis by mNGS. Therefore, it is advised to highlight the importance of RT-PCR in these situations due to its sensitivity and cost effectiveness than mNGS.
9. In section 3.3.1, line 509 needs to clarify the type of blood samples tested in the cited study. Whole blood or plasma cell free DNA extracted from blood? This is important for the readers to identify type of blood samples used in mNGS.
10. In line 265, use of > 300 10⁶/L is correct but ensure consistency with other units used in the same section ie; mg/L or g/L
11. Different font types were found. Suggest checking throughout manuscript and correcting.
12. In 3.5.1. The authors discussed the high sensitivity of mNGS in detecting uropathogens. However, in urine specimens in addition to uropathogens, normal urinary flora with less clinical abundance may also be detected by the mNGS. Therefore, it is important to mention the criteria used in the cited studies to distinguish between a UTI and normal urinary flora. Also mention about the specificity of mNGS in detecting UTIs.
13. Additionally, UTIs are very common and comparatively easy to treat. However, performing mNGS is expensive. Therefore, in this review authors need to explicitly mention when it is appropriate to perform mNGS in UTIs.
14. In line 764, you’ve mentioned about Orientia tsutsugamushi and it is a systemic rickettsial infection, not a primary UTI pathogen. It is needed to clarify this to ensure that readers do not misunderstand.
15. One of the major challenges in UTIs is MDR organisms. Under blood stream infections, the authors mentioned AMR. It would be relevant to mention the detection of AMR pathogens in urine also and briefly mention how mNGS and tNGS used to detect resistance genes in urine specimens.
16. In 5.2.1, authors mentioned misinterpretation by laboratory personnel may lead to discrepancies. Suggest mentioning personnel or team who should review these results before the results are released to the patient such as microbiologists, physicians etc
17. The authors mentioned that most tests are Laboratory Developed Tests (LDTs). This is a barrier for smaller hospitals. Suggest to expand the implications of this.
18. Also, you have mentioned about semi-quantitative capabilities. However, NGS techniques are not good as qPCR for absolute quantification. This should be stated with caution since, NGS is not a replacement for absolute quantification provided by the gold standard qPCR for viral loads. Suggest to clarify the recommendation of NGS for pathogen discovery than precise viral load monitoring.

 

References

References need to be formatted according to journal requirements. Eg: organisms’ names are not in italics in references, some references are underlined etc.

 

 

General comments

1. Suggest to improve English language of the manuscript with the help of an English editing service.
2. Several typing and punctuation errors were found throughout the manuscript that should be corrected.
3. Different font types were noted in the middle of some paragraphs. Suggest to check fonts throughout the manuscript.
4. Scientific names of organisms must be written in Italics.
5. Suggest to format the manuscript strictly following the journal’s guidelines.

 

Author Response

Dear Editor,

Thank you for your kind letter regarding our submitted manuscript entitled " Metagenomic and Targeted Next-Generation Sequencing in Infectious Disease Diagnostics: Current Applications, Challenges, and Future Perspectives （Manuscript ID: 4033105） ". We would like to express our sincere thanks to you and the reviewers for the constructive comments. The manuscript has been revised according to your instruction and is now being resubmitted. Thanks for giving us the precious opportunity to publish this paper in diagonostics

Our point-by-point responses are provided below. All revised or supplementary new text is highlighted in blue font in the revised manuscript.

Yours sincerely,

Authors

 

 

Reviewer1：

1. Suggest changing the title as follows

 

“Metagenomic and targeted next-generation sequencing in infectious disease diagnostics: current applications, challenges, and future perspectives”

 

Response: We sincerely thank reviewer for this helpful suggestion which makes the title more precise. We have changed the title follow the reviewer’s comment. The revised title has highlighted in blue font (Page 1, Line 1-4).

 

Introduction

2. In line 48-50, the authors mention that microbiological culture requires 24 hours for pathogen growth but have not mentioned the time required for mNGS. Suggest mentioning the turnaround time for mNGS also explicitly highlighting its rapidity compared to culture method.

Response: We feel sorry for this confusion. We compared the turnaround time of mNGS and cultural methods in line 920-923 (“In terms of rapidity, compared to traditional culture methods that typically require 48-72 hours to obtain results (and even more than a week for fastidious bacteria such as mycobacteria), the average turnaround time for conventional mNGS is 24 hours “), however, we acknowledge that this clarification of mNGS turnaround time should be introduced in Introduction part. We have clarified the turnaround time of mNGS in line 77-79.

3. In addition to mNGS, introducing targeted NGS also would be beneficial for the authors.

 

Response: We agree with the reviewer that it is helpful to present the theme of this study. In the revised manuscript, we have introduced targeted NGS in introduction part (Page 2-3, Line 88-95).

 

4. In line number 84 to 92, technical challenges are mentioned. In addition to that, I suggest adding about the handling of commensal organisms

 

Response: Thank you for your detailed review and constructive comments. We acknowledge that we should mention challenges about commensal organisms. For handling of commensal organisms, false positives and false negatives still remain significant issues. Meanwhile, mNGS shows lower sensitivity for RNA viruses’ detection. We have address reviewers’ concern in the revised manuscript (Page 3, Line 97-98, Line 102-104) and discussed these specifically in 5.2.1.

 

 

 

Next-Generation Sequencing Technologies

 

In general, the authors have provided a good technical breakdown of sequencing methods.

5. In section 2.2, suggest mentioning about the limitation of detection of tNGS in comparison with the mNGS and the clinical reasons for choosing these two techniques at specific instances.

 

Response: We also appreciate your suggestion to describe the limitation of detection of tNGS in comparison with the mNGS. tNGS is suitable for known or limited pathogen spectra but has disadvantages in the detection unknown etiology of complex infections, atypical infections, mixed infections and so on. We have added the limitation of tNGS in the revised manuscript, clarified how to choose these two different techniques at specific instances in the revised manuscript (Page 5, Line 200-202).

 

6. In section 2.1.1, line 128-130, the authors should briefly acknowledge the limitations of RNA sequencing. Eg; complexity, cost, cold chain requirement etc. also

 

Response: Thank you for the careful review and thoughtful suggestion. We have acknowledged the limitations of RNA sequencing from the aspects of complexity, cost, cold chain requirement in the revised manuscript (Page 4, Line 147-149).

 

7. In section 2.1.2, it would be beneficial to mention how accuracy and error rates vary among SGS and TGS.

 

Response: Thank you for your careful review and thoughtful suggestion. We have clarified the accuracy or error rates from different platform among SGS and TGS in the revised manuscript (Page 4, Line 164-165, Line 170-172).

 

8. In section 2.2.1.2, mentioning these data in words may affect readability. It is better to use a table to highlight the importance of mNGS, hc-tNGS, and mp-tNGS techniques.

Response: Thank you for your kind suggestion which enhanced readability. We have added a table in the revised manuscript. We compare the technology, range, advantages, disadvantages and application scenarios of mNGS, hc-tNGS, and mp-tNGS(Table 1. Page 6).

 

9. Scientific names of organisms should be italicized throughout the manuscript. Eg. Streptococcus pneumoniae in line 258, 760, 764, 767 etc.

 

Response: We appreciate the reviewer’s careful evaluation of our work. We regret for these careless mistakes. We have carefully italicized scientific names of organisms throughout the manuscript. These italicized scientific names of organisms were highlighted in blue font.

 

10. In line 261-264, authors have mentioned regarding mNGS performance among a group of patients who received empiric antibiotic treatment. Considering the significance of this point, I suggest emphasizing more on the importance mNGS testing in patients who have exposed to antibiotics prior to hospital admission and showing culture-negative results.

 

Response: We completely agree with the reviewer’s comment that it is necessary to emphasize more on the importance of mNGS in patients who have exposed to antibiotics prior to hospital admission and showing culture-negative results. We have clarified the advantages of mNGS in this situation, and added correlative references in the revised manuscript (Page 9, Line 305-308, Line 313-316).

 

11. In line 284-286, authors state that detecting of low viral load in viral encephalitis by mNGS. Therefore, it is advised to highlight the importance of RT-PCR in these situations due to its sensitivity and cost effectiveness than mNGS.

Response: Thank you for the insightful comment and thoughtful suggestion. We understand we should emphasize the advantages of RT-PCR when detecting viral encephalitis (Page 9, Line 330-332).

 

12. In section 3.3.1, line 509 needs to clarify the type of blood samples tested in the cited study. Whole blood or plasma cell free DNA extracted from blood? This is important for the readers to identify type of blood samples used in mNGS.

 

Response: We apologize for our oversight. In fact, the researchers from the cited study tested blood plasma cell free DNA extracted from blood. We have clarified the type of blood samples in the revised manuscript (Page 14, Line 553-554).

 

13. In line 265, use of > 300 10⁶/L is correct but ensure consistency with other units used in the same section ie; mg/L or g/L

Different font types were found. Suggest checking throughout manuscript and correcting.

 

Response: Thank you for your kindly comments. We sincerely apologize for our mistakes that may cause misunderstanding. However, in the CSF report, “10⁶/L” is used for white-cell counts, and mg/L is the appropriate unit for protein testing, so we have retained these two units (Page 9, Line 304-305).

Meanwhile, we have carefully checked the font types and unified the different styles.

 

14. In 3.5.1. The authors discussed the high sensitivity of mNGS in detecting uropathogens. However, in urine specimens in addition to uropathogens, normal urinary flora with less clinical abundance may also be detected by the mNGS. Therefore, it is important to mention the criteria used in the cited studies to distinguish between a UTI and normal urinary flora. Also mention about the specificity of mNGS in detecting UTIs.

 

Response: Thank you for these important suggestions. We acknowledge it is important to mention the criteria used in the cited studies to avoid misunderstandings. We have clarified the criteria distinguish between a UTI and normal urinary flora in the sited studies (Page 18, Line 774-778, Page 19, Line779-783, Line 785-787).

We also clarified the specificity of mNGS in detecting UTIs to enhance the reliability (Page 18, Line 773; Page 19, Line 788).

 

15. Additionally, UTIs are very common and comparatively easy to treat. However, performing mNGS is expensive. Therefore, in this review authors need to explicitly mention when it is appropriate to perform mNGS in UTIs.

 

Response：Thank you for this comment. We apologize for the lack of clarity regarding the indications for mNGS in UTIs. In the revised manuscript, we have now clearly specified the appropriate indications for mNGS from five aspects :1. recurrent or complicated UTIs with negative cultures despite high clinical suspicion, particularly in patients with prior antibiotic exposure; 2. immunocompromised patients, suspected opportunistic or atypical pathogens; 3. suspected polymicrobial infections involving fastidious, anaerobic, or non-culturable organisms; 4. severe or life-threatening urinary tract infections requiring rapid etiological identification when conventional tests remain negative; 5. epidemiological or infection control investigations requiring comprehensive pathogen and resistance gene profiling (Page 19, Line 814-825).

 

16. In line 764, you’ve mentioned about Orientia tsutsugamushi and it is a systemic rickettsial infection, not a primary UTI pathogen. It is needed to clarify this to ensure that readers do not misunderstand.

 

Response: Thank you for your carefully review. In the revised manuscript we have added that the patient presented with UTI symptoms, not a primary UTI (Page 19, Line 805).

 

17. One of the major challenges in UTIs is MDR organisms. Under blood stream infections, the authors mentioned AMR. It would be relevant to mention the detection of AMR pathogens in urine also and briefly mention how mNGS and tNGS used to detect resistance genes in urine specimens.

 

Response: We appreciate your suggestion to include the detection of antimicrobial resistance (AMR) pathogens in urine specimens, paralleling the discussion on bloodstream infections. Accordingly, we have revised the manuscript to address this point in the revised manuscript (Part 3.5.2 NGS in Antimicrobial Resistance Detection; Page 19-20, Line 826-861).

 

18. In 5.2.1, authors mentioned misinterpretation by laboratory personnel may lead to discrepancies. Suggest mentioning personnel or team who should review these results before the results are released to the patient such as microbiologists, physicians etc

 

Response: Thank you for the constructive feedback and the valuable suggestion. We have mentioned the microbiologists and physicians’ review work which can help to avoid misinterpretation by laboratory personnel in the revised manuscript (Page 24, Line 1010-1012).

 

19. The authors mentioned that most tests are Laboratory Developed Tests (LDTs). This is a barrier for smaller hospitals. Suggest to expand the implications of this.

 

Response: Thank you for your valuable suggestion to expand the discussion on the implications of most NGS assays being classified as Laboratory Developed Tests (LDTs). We agree that this regulatory status presents a significant barrier, particularly for smaller hospitals. We have clarified the barriers such as expertise, financial burden in the revised manuscript (Page 25, Line 1035-1039).

 

20. Also, you have mentioned about semi-quantitative capabilities. However, NGS techniques are not good as qPCR for absolute quantification. This should be stated with caution since, NGS is not a replacement for absolute quantification provided by the gold standard qPCR for viral loads. Suggest to clarify the recommendation of NGS for pathogen discovery than precise viral load monitoring.

 

Response: Thank you for the careful review and the kind suggestion. We are sorry about the unclear description. At present, NGS is largely qualitative method, and it is still a considerable way off from being developed into a reliable quantitative or semi-quantitative tool. We acknowledge that qPCR can’t be replaced by NGS due to the absolute quantification capabilities of qPCR. We have clarified this in the revised manuscript (Page 25, Line 1016-1021).

Reviewer 2 Report

Comments and Suggestions for Authors

The authors in this review outlines the current clinical applications of metagenomic and targeted next-generation sequencing in infectious disease diagnostics. It highlights their value in pathogen identification, antimicrobial resistance and virulence profiling, and outbreak investigation, particularly in complex respiratory and critically ill patient settings. The review also discusses key technical and practical limitations, including analytical performance, standardization, bioinformatic complexity, and costs, and explores future perspectives for integrating NGS-based surveillance into routine clinical microbiology and public health practice.
The review is informative, however requires a minor adjustment concerning several points throughout the manuscript. See the comments in the attached file. 

Comments for author File: Comments.pdf

Author Response

Dear Editor,

Thank you for your kind letter regarding our submitted manuscript entitled " Metagenomic and Targeted Next-Generation Sequencing in Infectious Disease Diagnostics: Current Applications, Challenges, and Future Perspectives （Manuscript ID: 4033105） ". We would like to express our sincere thanks to you and the reviewers for the constructive comments. The manuscript has been revised according to your instruction and is now being resubmitted. Thanks for giving us the precious opportunity to publish this paper in diagonostics

Our point-by-point responses are provided below. All revised or supplementary new text is highlighted in blue font in the revised manuscript.

Yours sincerely,

Authors

 

 

 

Reviewer2：

 

Premise:

The comments take into account the structure and tone of the review, which appears to

be intended for reading by clinical staff.

 

1. The structure of the review, in which each body district is discussed separately might make it difficult to grasp all data presented. Please consider adding a table comparing different aspects of tNGS vs mNGS, such as:

– TaT

– Costs

– Sensitivity

– Targets covered

– A priori knowledge of pathogens needed

– Bioinformatic analysis complexity

 

Response: Thank you for your helpful suggestion. We have made a table to compare mNGS and tNGS (Table 4, Page 24). We must clarify that due to differences in study populations and the pathogens detected, the sensitivities of mNGS and tNGS vary across different scenarios. Therefore, we have provided separate tables (Table 2&3 Page 22-23) to present the sensitivities of both methods and discussed this point in the revised manuscript (Page 24, Line 989-990).

 

2. Lines 119-120:

“mNGS not only identifies [...] their genetic makeup” seems to be addressing 2nd/3rd level results to the mNGS technique, which is true in a way, but could be better conveyed expliciting that it provides “unbiased” data about the sample composition and state at the moment of sequencing.

Response: We appreciate your careful evaluation of our work. We have rephased this sentence to clarified that mNGS can provide unbiased data in the revised manuscript (Page 3, Line 134-138).

 

3. Lines 121-130:

Please rephrase the following sentences

123: “sequencing comparison process”

125-130: rephrase and explain more clearly. What does “testing” mean? QC?

Extraction? Sequencing?

Response: Thank you for pointing out the incorrect wording. We have rephased “sequencing comparison process” into “sequence alignment process” in the revised manuscript (Page 4, Line 141).

For “testing”, we sincerely thank the reviewer for highlighting this lack of clarity. We apologize for the confusion in the original manuscript. The “testing” here equals to DNA or RNA sequencing. We have rephased the description about RNA and DNA sequencing in the revised manuscript (Page 4, Line 146, Line 149).

4. 132: Substitute “the samples can be” with “being processed”.

Response: We sincerely appreciate the reviewer’s carefully evaluation. We have substituted the phrase according to the reviewer’ s advice. The revised sentence was highlighted in blue font (Page 4, Line 153).

 

5. 135: Change “are the same for different samples” to “can be the same for different

samples”, since it is not always true.

Response: We sincerely appreciate the reviewer’s carefully evaluation and serious attitude. We have substituted the phrase according to the reviewer’s advise. The revised sentence was highlighted in blue font (Page 4, Line 156).

 

6. 145-147:This sentence is missing prior discussion about amplification vs native DNA/RNA sequencing. It doesn’t really fit at the moment, please expand the subject or give prior informations about it.

 

Response: Thank you for your careful review and helpful advice. In the revised manuscript, we have added prior information about SGS and TGS in the introduction part (“mNGS is typically detected using second-generation sequencing (SGS) or third-generation sequencing (TGS) platforms, representing a transformative diagnostic paradigm that addresses many of these limitations.” Page 2, Line 75-77). Follow the reviewer’s suggestions, we rephased part 2.1.2. Technological Platforms: SGS vs. TGS. We expand the introduction of SGS and TGS from the aspects of accuracy, bias, and platform, to make the sentences readable and fit at the moment (Page 4, Line 159-177).

 

7. 154: Please rephrase “sequencing dataset”.

 

Response: Thank you for pointing out the incorrect wording. We have rephased “sequencing dataset” into “target panel” in the revised manuscript (Page 5, Line 184).

 

8. 176: “clinical expert consensus documents” – what did you mean? Please rephrase.

 

Response: Thank you for pointing out this issue. We sincerely apologize for this oversight. In fact, “clinical expert consensus documents” is a type of published literature. To avoid ambiguity, we have rephrased this statement in the revised manuscript (Page 7, Line 211-213).

 

9. 178-179: “A typical [...] 200 target species” – source?

Response: Thank you for your careful review, we sincerely apologize for this mistake. The source is:

“Wang Y, Wang H, Li Y, et al. Analysis of pertussis surveillance data (2022-2023) in a district of Beijing. BMC Infect Dis. 2025;25(1):1834. Published 2025 Nov 28. doi:10.1186/s12879-025-12225-2.”

We have added this reference in the revised manuscript (Ref 29. Page 7, Line 211-213).

10. 180: “Reference databases [...] RefSeq [24]” – Is it the only database used? Are the

other DBs actually checked as well? Please give at least 1 more example and add

1/2 references at other databases.

 

Response: Thank you for your insightful suggestion regarding the references. We completely agree that citing relevant databases the manuscript and properly contextualizes our findings. Following the reviewer’s advice, we have added several key databases (GenBank, Comprehensive Antibiotic Research Database, CARD, Virulence Factors Database, VFDB) to support our claims in the revised manuscript (Page 7, Line 214, Line 216-219).

 

11. 252: Please give a minimum of context/explanation to what STARD is.

Response: Thank you for pointing out the omission, we apologize for not defining “STARD” clearly in the original manuscript. STARD stands for “Standards for Reporting of Diagnostic Accuracy Studies”. Following the reviewer’s suggestion, we have added the full term and a brief explanation in the revised manuscript (Page 8, Line 288-289).

 

12. 266-267: “Sequencing [...] monitoring.” – Phrased like this, I understand that the number of total reads increase with inflammatory state. Did you mean it? Or perhaps, certain reads, which can be assigned to certain cell types/pathogens increase in proportion? Please rephrase and improve explanation.

 

Response: Thank you for your carefully review. We agree with the reviewer that while the proportion of pathogen-specific reads may increase with inflammation. Follow the reviewer’s suggestion, we have rephrased the statement to avoid misunderstanding (Page 9, Line 308-310).

 

13. 286: “underrepresentation of RNA sequencing” – what do you mean? Please rephrase.

 

Response: Thank you for your carefully review. We apologize for the incorrect wording. Here we want to explain that the degradability of RNA is one of the limitations of mNGS. We have changed “underrepresentation of RNA sequencing” into “the degradability of RNA sequencing” in the revised manuscript (Page 9, Line 332).

 

14. 373-376: This sentence is ok, but the message is a bit redundant throughout the text, please consider using it less frequently.

Response: Thank you for your helpful suggestion, we have restructured the sentence to make it more concise in the revised manuscript (Page 9 Line 419-421).

 

15. 431-435: See above comment. At this point of the reading, such sentences start feeling redundant.

 

Response: We appreciate the reviewer’s constructive suggestion. We have rephrased sentences in the revised manuscript (Page 12 Line 483-484).

 

16. 443-448: See the two comments above.

 

Response: Thank you for your valuable comments. These sentences are conclusions for the whole paragraph (mNGS in Novel Pathogen Detection; Page 12 Line 472-487). Therefore, in the revised manuscript, we rephrased the sentences to make them more concise and precise, rather than delete them. (Page 12 Line 483-484).

      

17. 430+436-437: You could collapse the discovery of Sars-CoV-2 in a single piece.

 

Response: Thank you for your valuable comments. We have collapsed the discoveries in the revised manuscript (Page 12 Line 474-478).

 

18. 509-512: Please rephrase, you first introduce a certain number of patients, then talk about specimens and then again patients. Make the sentence more consistent, both regarding numbers and terms.

 

Response: Thank you for your kind comments. We sincerely apologize for the oversight that makes the manuscript confused. We have revised the numbers and terms in the revised manuscript (Page 14, Line 553-556).

 

19. 506: Regarding chapter 3.3 (starting page 11), please consider removing “common” from the first subheading and merge sub-chapters 1 to 4 (bacterial, viral and fungal infections). It might reduce redundancy of concepts and sentences, yielding an easier reading experience.

Response: Thank you for your helpful suggestions that improve the quality of our manuscript. We have deleted “common” and merge sub-chapters 1 to 4 (bacterial, viral and fungal infections) to make the expression clearer in the revised manuscript (paragraph mNGS in Bloodstream Infections, Page 14-15, Line 552-608).

 

20. 552-554: Please rephrase and be more clear.

 

Response: Thank you for your helpful suggestion. We have rephrased the sentence to make it clearer in the revised manuscript (Page 15, Line 591-593).

 

21. 580: Please check if cfDNA was already introduced as abbreviation. Maybe even give a hint of what cfDNA is. Additionally, there is a missing (or additional) parenthesis.

Response: We apologize for the confusion. We have introduced cfDNA in line 553 and clarified it can’t be identified by conventional tests in line 586-587. We apologize for incorrectly using parentheses. We have deleted parenthesis in the revised manuscript (Page 15, Line 621).

 

22. 581: “when applied to cfDNA from 1,250 clinically [...] alert patients” – would be better phrased as “when applied to cfDNA from 1,250 sepsis alert patients demonstrated high sensitivity and specificity towards...”

Response: Thank you for your kind suggestion. We have rephrased the sentence according to your advice in the revised manuscript (Page 15, Line 622-624).

 

23. 631:This sentence is correct, but it could also be applied to mNGS – please point that out.

Response: Thank you for your carefully review. We have pointed out that both tNGS and mNGS are able to detect rare or mixed infections, the revised expression was highlighted in blue font (Page 16, Line 667-668).

 

24. 726-735: I noticed that the message of this paragraph is repeated throughout every chapter. Was it intentional? Maybe for M.D.s that would read only chapters regarding their speciality? If it was not by design, please reduce redundancy.

 

Response: Thank you for your carefully review. However, writing approach here is to demonstrate that NGS can identify antibiotic-resistant bacteria and mixed infections across different systems and types of infections caused by various modes of infection (e.g. Respiratory System Infections, Bloodstream Infections, Digestive System Infections), thereby highlighting its powerful capabilities. We think it’s our incorrect expression results the misunderstanding and we sincerely apologize for it. Therefore, we would like to rephrase and retain these sections in the revised manuscript (Page 18, Line 761-766).

 

25. 836: Please explicitly state that 6hrs tat can be achieved in dedicated and well organized contexts.

 

Response: We appreciate the reviewer’s advice that enables us to express opinion more precisely. We have clarified the prerequisite of 6hrs tat follow the advice (Page 22, Line 923-924).

 

26. 843-857: Please rephrase; Summarizing the previous chapters in this way risks conveying the message that certain advantages are specific to each district or body tissue, whereas they are transversal.

 

Response: Thank you for this insightful suggestion. Our original summary inadvertently implied that certain advantages of mNGS might be specific to individual districts or body tissues, leading to this ambiguity. Your opinion is absolutely correct, these advantages, such as rapid, unbiased pathogen detection, high sensitivity, are transversal. To address this, we have carefully rephrased the summary section in the revised manuscript (Page 22, Line 931-938).

 

27. 864: Please introduce coverage and depth subjects prior to utilizing them in the text; M.D.s and people not in the genomic field would not understand their importance and usefulness, you should not give them for granted. Please, explain how depth can impact clinical sensibility and pathogen(s) discrimination.

 

Response: We thank the reviewer for this valuable suggestion regarding the introduction of sequencing depth and genomic coverage. In revised manuscript, we have now added the section to provide clear definitions of sequencing depth and genomic coverage, explains their clinical relevance, including how depth impacts sensitivity for low-abundance pathogen detection and how coverage enables accurate pathogen discrimination (Page 23, Line 947-964).

 

28. 867: “simpler workflows” – is not an absolute truth; certain targeted protocols are for sure more laborious than shotgun/metagenomic ones.

 

Response: Thank you for this important correction. Your opinion is absolutely right. In fact, as the reviewer points out, certain targeted protocols of tNGS can be more laborious than shotgun/metagenomic approaches due to additional steps such as multiplex PCR setup and amplicon processing. We have deleted “simpler workflows” and rephrased this statement to avoid overgeneralization (Page 23, Line 967-969).

 

29. 911-913: Please give context of single/low number of samples vs batch sequencing.

 

Response:Thank you for this insightful comment. We have added contextual explanation regarding the operational distinction between single-sample ("on-demand") and batch sequencing modes in the revised manuscript (Page 25, Line 1023-1032).

 

30. 913: Please add context to metabolic pathway usefulness. They were not introduced

previously and are a bit out of context.

 

Response: Thank you for your careful review. The original sentence “sequencing depth may not always be sufficient to accurately predict metabolic pathways for individual species, especially in samples with low biomass or those contaminated with host DNA” comes from hi Y, Wang G, Lau HC, Yu J. Metagenomic Sequencing for Microbial DNA in Human Samples: Emerging Technological Advances. In fact, this article didn’t explain the relation between metabolic pathways and infection. We apologize for our incorrect statement and thank the reviewer for helping us improve the clarity and focus of the manuscript. After re-reading this section, we agree that predicting metabolic pathways does not align well with the main focus of our discussion and was not adequately grounded in the data or prior introduction. Rather than adding superficial context that might further distract from the core message, we have decided that the most appropriate course of action is to remove this sentence entirely.

 

31. 916: Please change “with most currently” to “being mostly”

 

Response: Thank you for the careful review and the thoughtful suggestion. We have changed the sentence and highlighted it in blue font (Page 25, Line 1034).

 

32. 923-925: It is true (maybe even more true) for mNGS too.

Response: Thank you for your insightful advice. Your opinion is absolutely right. We have clarified that mNGS aslo is restricted by such limitations in the revised manuscript (Page 25, Line 1049-1050).

 

33. 938-940: See previous comment.

Response: Thank you for your insightful advice. Your opinion is absolutely right. In the revised manuscript, we have clarified that mNGS is also under the same situation (Page 26, Line 1063-1064).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for taking time to edit the manuscript based on comments, the article can be accepted for publication in its current format