Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Mthembu and colleagues, in the manuscript entitled "Adapting The Illumina COVIDSeq for Whole Genome Sequencing of other Respiratory Viruses in Multiple Workflows and a Single Rapid Workflow," efficiently described a new method to help in the detection and characterization of respiratory viruses. The authors compared the classical method with the new one that they developed.

The comparison is well described, and each phase was accurately compared between the two methods. 

This paper will help on the future use of NGS techniques for the detection and characterization of respiratory viruses, but also for other viral types.

In my opinion the manuscript could be published, paying attention to some, minor revision, listed below:

1. Please write the names of viruses in normal characters; they are not bacteria.
2. The ethical statement should be added at the end of the manuscript, in the "informed consent statement."
3. Figures 1 and 2 appear redundant. Try to fuse the figures into a single one, highlighting the differences in phase 3.

Author Response

Comment 1: Please write the names of viruses in normal characters; they are not bacteria.

Thank you for taking your time to review our work and for the constructive feedback. Viruses' names have been updated accordingly on the revised manuscript, in lines 31-33, 45, 54-56, 73-74, 81, 84, 98, 105-107, 124-126, 146, 198-199, 248, 252-257, 259-260, 323-325, 374, 392,404-406, 482-483

 

Comment 2: The ethical statement should be added at the end of the manuscript, in the "informed consent statement.

Thank you for the constructive feedback. Informed consent has been updated on the revised manuscript, in lines 470-476.

"Institutional Review Board Statement: All protocols were reviewed and approved by the Biomedical Research Ethics Committee (BREC) of the University of KwaZulu-Natal (protocol reference number: BREC/00007092/2024), valid between 14 July 2024 and 13 July 2026.

Informed Consent Statement: Informed consent for participation was not required as per South Africa’s Department of Health Ethics in Health Research Guidelines (March 2015), Section  3.3.5 and Section 3.3.6."

 

Comment 3: Figures 1 and 2 appear redundant. Try to fuse the figures into a single one, highlighting the differences in phase 3.

Thank you, this is extremely helpful, the figure has been revised accordingly on line 180. 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Revision Report

 

"Adapting The Illumina COVIDSeq for Whole Genome 2 Sequencing of other Respiratory Viruses in Multiple 3 Workflows and a Single Rapid Workflow"

Manuscript number: labmed-3728696

 

Rationale:

The manuscript evaluated the possibility of adapting the Illumina COVIDSeq assay—originally designed for SARS-CoV-2 whole-genome sequencing—for use with other respiratory viruses such as Influenza A/B, Respiratory Syncytial Virus (RSV), and Rhinovirus. The authors used positive control samples from the aforementioned viruses and tested two approaches for library preparation: one is four (4) virus-specific multiple workflows, and a combined rapid workflow. The results show that the COVIDSeq assay can be effectively adapted for multi-pathogen genomic surveillance, and that the combined rapid workflow can offer a cost- and labor-efficient alternative.

General comments:

The manuscript is highly informative and exceptionally well-written. The authors present their ideas effectively, and the description is clear and concise. Additionally, acknowledging the study's limitations in the discussion is both important and well addressed.

The main points need to be taken care of

1. Figure 1 and 2; should be combined as A and B. This makes the idea clearer and straightforward,

 

1. In the section “Comparison Cost of Quality Control Reagents between the Rapid and the Multiple Workflow”. Please specify the date when these prices were obtained. Additionally, clarify that the cost calculations are based on combining the 14 samples.

Author Response

Comment 1: Figures 1 and 2 should be combined as A and B. This makes the idea clearer and straightforward,

Thank you for taking the time to review our work. This is extremely helpful. The Figure has been revised accordingly on line 180.  

 

Comment 2:  In the section “Comparison Cost of Quality Control Reagents between the Rapid and the Multiple Workflow”. Please specify the date when these prices were obtained. Additionally, clarify that the cost calculations are based on combining the 14 samples.

Thank you very much for the feedback, it has been adjusted according to line 271-291.

"3.5 Comparison of the Cost of Quality Control Reagents between the Rapid and the Multiple Workflow in USD

The cost comparison of quality control reagents between the rapid and multiple workflows in Table 3 below shows a reduction in the cost of quality reagents between the two approaches, as the rapid workflow library preparation results in one library and the multiple workflow with four libraries. The TAKARA Library Quantification Kit, priced at $972.54 (500 reactions), as of the 11^th of March 2025, resulted in $1.94 for the single library with the rapid workflow approach and $7.78 for the four libraries using the multiple workflows approach. The Qubit 1X dsDNA HS Kit, costing $102.59 (100 reactions), as of the 20^th of February 2025, contributed $1.03 for the single library, the rapid workflow,$4.10 for four libraries in the multiple workflow. For the E-Gel EX Gels (1%), with a kit cost of $119.05 (10 packs), as of 21^st of February 2025, the rapid workflow incurred $11.91 for the single library, compared to $47.62 for four libraries in the multiple workflow. Overall, the total quality control cost per sample was $14.88, processing 7 samples for the rapid workflow and $59.50 processing 7 samples for the multiple workflow, highlighting a more cost-effective solution with the rapid method."