Review Reports - Ct Value from RT-qPCR Can Predict SARS-CoV-2 Virus Assembly and Lineage Assignment Success

Round 1

Reviewer 1 Report

The main idea of the article is: if you take samples with lower Ct values, then the assembly of genomes will be better. There is no scientific novelty in this statement. It is clear that the lower is Ct, the greater is the initial concentration of the virus in the sample. It is equally clear that the more RNA in the sample gives the better sequencing and assembly. This fact is widely known, and when sampling for sequencing, everyone evaluates Ct.

In accordance with the foregoing, I believe that the article has no scientific novelty and do not recommend this article for publication.

Author Response

Reviewer's comment:

In accordance with the foregoing, I believe that the article has no scientific novelty and do not recommend this article for publication.

Our reply:

Dear Reviewer,

thank you for reading our article and providing your opinion. Although connection between lower Ct values and assembly quality through more viral load, then more viral RNA to sequence -> better coverage -> better assembly would be very logical and sounds like obvious and we understand reviewer’s reasoning, however it seems it is not easy to find paper about direct connection between RTq-PCR Ct values, especially for experimental design like ours. We are not stating that no one noticed this fact before, but our article is showing actual relationship between assembly metrics and RT-qPCR Ct values under experimental design for metatranscriptomic study from human tissue (instead of standard design with isolating viral RNA). Our article acknowledged, that it is possible to predict assembly success by actual RT-qPCR Ct values directly. Maybe problem was not stating the facts of the specifics for our study design clearly enough. To address this issue, we added our argumentation to discussion section and added or changed few sentences in text to improve clarity and hoping for second consideration. Thank you.

Reviewer 2 Report

To monitor the spread and emergence of SARS-CoV-2 new genetic variants, the viral genome sequences from high throughput next generation sequencing techniques are necessary. Hadzega and colleagues in their manuscript demonstrated that the Ct-value from the RT-PCR diagnostic tests can predict virus assembly and lineage assignment success. Their findings are clear and based on the presented data. The manuscript is well written and easy to follow, but still I have some suggestions for changes, please see below.

MAJOR COMMENT

1. I would recommend to change the Ct value to the viral load, since the Ct value may depend on the RT-PCR diagnostic test manufacturer.

MINOR COMMENTS

A. Please adjust the citation format to the MDPI recommendations.

B. It would be good to add some conclusion at the end of the abstract.

C. The number of patients is really misleading. How many samples from patients with severe and mild COVID-19 were collected? How many samples did you collect from patients with suspected COVID-19? What are the controls from whom nasopharyngeal swabs were collected?

Author Response

Dear reviewer,

thank you for reading our article and providing your suggestions, comments and valuable feedback. Here you can find our answers and reactions for your questions and comments.

MAJOR COMMENT

I would recommend to change the Ct value to the viral load, since the Ct value may depend on the RT-PCR diagnostic test manufacturer.

Thank you for your thoughtful feedback on our paper. We appreciate your suggestion to change the Ct values to viral load. We would like to offer the following rationale for our decision to use Ct values in our analysis. Our study exclusively utilized an RT-PCR kit from a single manufacturer. While your recommendation to convert Ct values to viral load is indeed valid in cases where different RT-PCR kits are used, our study design centered around a consistent approach using one kit. As such, the potential variations due to different manufacturers are minimized within our dataset. We acknowledge the importance of viral load quantification for a comprehensive understanding of the data. However, our study is retrospective in nature, and generating a reliable standard curve for conversion retrospectively presents challenges. The availability of samples with known viral loads, necessary for calibration curve construction, was not accounted for in our original design. Utilizing Ct values across all samples ensures a consistent dataset for analysis and interpretation. Converting Ct values to viral load would introduce an additional layer of complexity that may not align with the scope and goals of our study. Moreover, given the single manufacturer source, our analysis retains a relative consistency in Ct values that can still provide insights into the trends and patterns we are investigating. We appreciate the value of reporting viral load directly, and while we acknowledge this limitation, we believe that our approach of using Ct values maintains internal consistency within our dataset. We are committed to providing a transparent discussion in the manuscript that highlights this limitation and contextualizes the significance of our results within the scope of our study's objectives and constraints.

MINOR COMMENTS

Please adjust the citation format to the MDPI recommendations.

Thank you for your notice. We are aware of incorrect citation format. We changed the citation style. If there are still incorrect details in format, we will certainly correct them after approval of the paper.

B. It would be good to add some conclusion at the end of the abstract.

Thank you for your suggestion. We added short conclusion to the abstract.

The number of patients is really misleading. How many samples from patients with severe and mild COVID-19 were collected? How many samples did you collect from patients with suspected COVID-19? What are the controls from whom nasopharyngeal swabs were collected?

Thank you for your questions. We provided number of patients with severe symptoms, those with mild symptoms and number of asymptomatic samples (all added at the methods section). We added information about negative controls (samples at methods section) and result of their assembly attempt (results section).

Reviewer 3 Report

Hadžega et al. presented the results of a comparative study on SARS-CoV-2 genome assembly and lineage assignment in relation to the Ct values of collected clinical samples. While this parameter is crucial for effectively managing clinical samples for both diagnostic and research purposes, the novelty of the paper is limited. It is well-known that higher viral loads (lower Ct values) yield better results in virus identification and assignment, regardless of the sequencing strategy employed. Additionally, the authors compare their findings with data from Xiao et al.

The previous work by Xiao et al. also explored the presence of viral and/or bacterial co-infections in selected samples. However, this aspect was not addressed in the study by Hadžega et al. Therefore, drawing a comparison with previously published reports or stating that the newly developed system is suitable for metatranscriptomic analysis might be inappropriate.

I apologize for any misunderstanding, but the intended target of the presented manuscript seems somewhat unclear to me. Was the sole expectation focused on virus genome assembly and lineage assignment, or did it also encompass microbiome analysis? If the latter was indeed included, I couldn't find any provided data to support such a conclusion. However, following are my concerns regarding the manuscript.

Major comments

· Lines 29-34: Some mutations were described in terms of nucleotide sequences, while others were discussed at the protein level. For consistency, it is advisable to standardize the presentation. In my opinion, emphasizing mutations at the protein level would be more appropriate, considering that nucleotide variations can result in missense changes.

· Lane 71: “suspected of having COVID-19”. Since you studied SARS-CoV-2 genome, the enrolled samples were supposed to be positive for the virus. Please, comment or rephrase.

· Lanes 78-79: “Nasopharyngeal swabs specimens were collected from COVID-19 patients and controls and stored in viRNAtrap collection medium (GeneSpector, Czech Republic) at 4°C”. When were the samples collected? As indicated in paragraph 3.2, the presence of Alpha and Delta variants was detected. Given that these variants are no longer in circulation, it is expected that the samples were either stored at -80°C or promptly processed at the time of collection. Please provide your comments on this matter.

· Lanes 82-83: Were all the RNA extracts quantified using Qubit? Additionally, does the extraction kit employed involve the use of carrier RNA to supplement the samples? This information would be valuable to ascertain, particularly considering that a significant portion of the quantified nucleic acid by Qubit might be attributed to the carrier.

· Lanes 96-98: What was the number of samples pooled per run? This detail is crucial for enhancing the count of specific reads and consequently improving virus genome coverage, particularly if carrier RNA was included in the extract and also indexed. I would appreciate your comments on this aspect

· Lines 139-140: “assembly was fragmented in 2 or more scaffolds, but its alignment covered almost the whole reference genome”. What was the length of these two scaffolds? Were those overlapping? What was the genome coverage? Please, comment.

· Lane 151: it would be of interest to know the Ct range of each group or, at least, provide a mean Ct ± SD or the variance.

· Lines 215-221: Were other extraction methods tested? gDNA and rRNA depletion best fit with metagenomic and transcriptomic analysis. Please, comment on that in the discussion.

Minor comments

· An English spelling check is recommended for the purpose of rephrasing unclear sentences.

· Did the authors evaluate the correlation between sequencing performance and other viral targets' Ct values (e.g., N or RdRp)?

· Lanes 42-44: “When it comes to the routinely used methods utilized in standard 42 clinical sample processing during this pandemic mostly targeted enrichment strategies 43 were used, based on either amplicon or hybridization-probe approaches.”. Please, reframe.

Comments for author File: Comments.pdf

An English spelling check is recommended for the purpose of rephrasing unclear sentences.

Author Response

Reviewer's comments:

Our Reply:

Dear reviewer, thank you for reading our article and providing your suggestions, comments and valuable feedback. Here you can find our answers and reactions for your questions and comments. To address your questions and concerns, we provide following answers.

We mentioned work of Xiao et al. in purpose to compare specific result - viral load or Ct value, that suffice for succesefull genome construction and further analysis. We do not compare with other results of mentioned study with our results. Although bacterial genetic material was also studied, it is not part of this paper, but now it is already published as separate paper - Hyblova et al., 2023 (doi:10.3390/microorganisms11071804). Intention of our study was to show, that it is possible to predict assembly success of viral (specificaly SARS-COV2, but it should be similar with other viruses) genome from metatranscriptomic sequencing using just Ct value from RT qPCR. We did some changes to manuscript, hopefully it might be clearer now.

Major comments

Lines 29-34: Some mutations were described in terms of nucleotide sequences, while others were discussed at the protein level. For consistency, it is advisable to standardize the presentation. In my opinion, emphasizing mutations at the protein level would be more appropriate, considering that nucleotide variations can result in missense changes.

Thank you for your comment. It is very reasonable advice to standardise the presentation. Although nucleotide mutation might have effect without changing aminoacid in a protein in some cases (not claiming in this case), so it seems it is not simple issue to choose what is better for presenting, we take your advice and change the text (in case of variant alpha, that was presented in nucleotide mutantions). However it might not be possible to present it completely clearly, since we tried to mention it in short and reality of it is more complex.

Lane 71: “suspected of having COVID-19”. Since you studied SARS-CoV-2 genome, the enrolled samples were supposed to be positive for the virus. Please, comment or rephrase.

Thank you for your comment. We have revised the terminology to 'patients showing COVID-19 symptoms.' Initially, we used the term 'suspected' because the positivity for COVID-19 was later confirmed through RT-qPCR, making the samples known to be positive. The use of 'suspected' was intended to indicate the patients' perspective, reflecting their condition prior to the sequencing process. With this adjustment, we believe the sentence now provides greater clarity in describing the origin of the samples

Lanes 78-79: “Nasopharyngeal swabs specimens were collected from COVID-19 patients and controls and stored in viRNAtrap collection medium (GeneSpector, Czech Republic) at 4°C”. When were the samples collected? As indicated in paragraph 3.2, the presence of Alpha and Delta variants was detected. Given that these variants are no longer in circulation, it is expected that the samples were either stored at -80°C or promptly processed at the time of collection. Please provide your comments on this matter.

Thank you for your question. Nasopharyngeal swabs were collected from March 2021 to October 2022 in viRNAtrap transport medium (GeneSpector Innovations, Prague, Czech Republic), i.e. during the period of occurrence of all significant virus strains from alpha to omicron. RNA was isolated retrospectively from a nasopharyngeal swab-originated collection. RNA isolates were stored at -80 °C until processing into a genomic library and sequencing.

Lanes 82-83: Were all the RNA extracts quantified using Qubit? Additionally, does the extraction kit employed involve the use of carrier RNA to supplement the samples? This information would be valuable to ascertain, particularly considering that a significant portion of the quantified nucleic acid by Qubit might be attributed to the carrier.

Thank you for your question. RNA was isolated using the Cytiva® Sera-XtractaTM virus/Pathogen Kit (Global Life Sciences Solutions Operations, Little Chalfont, UK), automated magnetic bead-based procedure without using carrier RNA, so this was not an issue. All RNA samples after isolation were measured fluorometrically using QubitTM RNA High sensitivity (Invitrogen, Eugene, Oregon, USA).

Lanes 96-98: What was the number of samples pooled per run? This detail is crucial for enhancing the count of specific reads and consequently improving virus genome coverage, particularly if carrier RNA was included in the extract and also indexed. I would appreciate your comments on this aspect

Thank you for your question Paired-end sequencing (2 × 75 and 2 × 100) was performed on NextSeq500/550 and NextSeq2000 platforms, respectively. Samples were pooled at 24 per run and the mean number of read pairs per sample after trimming was 45.3 M (27.7–133 M) which can be considered sufficient for metatranscriptome sequencing.

Lines 139-140: “assembly was fragmented in 2 or more scaffolds, but its alignment covered almost the whole reference genome”. What was the length of these two scaffolds? Were those overlapping? What was the genome coverage? Please, comment.

Thank you for your questions. In all of the “fragmented” samples, those fragments cover around 99% of the reference genome. Number of fragments varies from 9 to 39, as well as fragments length from hundreds of bases to thousands. For the purpose of evaluating quality and visualising it, we used NG50 value. This way we believe it makes it simpler for a reader. Of course, we can provide the actual lengths of the scaffolds if needed. Regarding overlapping question, from visualization of alignments with reference and each contig to each by mummer and nucmer tools, assembled scaffolds appear to be unconnected fragments covering the reference genome without vast overlappings between each other. This is except one case, when two scaffolds of full length were assembled, both covering practically whole reference genome.

Lane 151: it would be of interest to know the Ct range of each group or, at least, provide a mean Ct ± SD or the variance.

Thank you for your comment. We added Table 1 to provide suggested statistics.

Lines 215-221: Were other extraction methods tested? gDNA and rRNA depletion best fit with metagenomic and transcriptomic analysis. Please, comment on that in the discussion.

Thank you for your question. In our study, we utilized the Kappa HyperPrep with RiboErase Kit (Kapa Biosystems, Salt River Cape Town, South Africa) for library preparation, incorporating eukaryotic RNA depletion for 18S rRNA as guided by the manufacturer's recommendations. It's important to note that our choice of extraction method was based on its compatibility with our objectives and our assessment of its suitability for metagenomic and transcriptomic analysis. We acknowledge that various extraction methods, including those involving gDNA and rRNA depletion, are frequently employed for metagenomic and transcriptomic analyses due to their ability to enhance the sensitivity of microbial and viral detection. However, in the specific context of our study, our primary focus was on evaluating the active microbiome sequences, including the viral component, rather than an exhaustive comparison of different extraction techniques. While we did not explore alternative extraction methods in this study, we will incorporate a discussion in our manuscript to elaborate on the advantages and limitations of the chosen extraction method and its implications for metagenomic and transcriptomic analyses. This will help provide context for the readers and researchers who are interested in the broader aspects of extraction methodologies in the context of microbiome studies.

Minor comments

An English spelling check is recommended for the purpose of rephrasing unclear sentences.

If needed we will send the article to professional spell-check.

Did the authors evaluate the correlation between sequencing performance and other viral targets' Ct values (e.g., N or RdRp)?

All RNA samples underwent RT-PCR analysis to determine the presence or absence of SARS-CoV-2 using the COVID-19 Real-Time Multiplex RT-PCR Kit (Labsystems Diagnostics, Vantaa, Finland). This testing was conducted on the ABI QuantStudio 6 Real-Time PCR System RT-qPCR platform (ThermoFisher Scientific, Waltham, Massachusetts, USA) following the original manufacturer's protocols. The kit employed gene targets ORF1ab, N, and E, in addition to an internal control (IC). A Ct value of less than 40 was considered indicative of positivity for at least one target, and it's noteworthy that the Ct values demonstrated consistency and similarity across all three targets (ORF1ab, N, and E). However, it's customary to report the Ct value specifically only for the E gene. Thus, for the purpose of our study, we utilized the Ct values from the E gene. It's important to mention that adopting Ct values from other gene targets (ORF and N) would not alter the outcomes. In fact, correlation coeficients (comparing N or ORF1ab gene Ct value with assembly outcome) was very similar to the one with E gene Ct value.

Lanes 42-44: “When it comes to the routinely used methods utilized in standard 42 clinical sample processing during this pandemic mostly targeted enrichment strategies 43 were used, based on either amplicon or hybridization-probe approaches.”. Please, reframe.

During the pandemic, standard clinical sample processing often involved the utilization of commonly employed methods. These methods primarily focused on targeted enrichment strategies, which were based on either amplicon or hybridization-probe approaches.

Round 2

Reviewer 1 Report

Dear authors, despite your additions to the text, the essence of the article has not changed and the described fact still seems to me quite obvious and the novelty of the paper is still limited.

Author Response

Dear reviewer,

since you did not comment on anything that was not discussed on previous review, we can only thank you again for reading our manuscript and providing your opinion.

Reviewer 3 Report

Authors fully responded to my criticisms and comments on the previously submitted manuscript.

Author Response

Dear reviewer,

We are glad you were satisfied with our answers. Thank you again for reading our manuscript and providing us with the valuable insight.