A High-Throughput Absolute Abundance Quantification Method for the Characterisation of Daqu Core Fungal Communities
Round 1
Reviewer 1 Report
A high-throughput absolute abundance quantification method for the characterisation of core fungal communities
It is interesting approach for newly developed method. It could be used widely for quantification of fungal communities, maybe, it is not clearly explained in paper.
The work, as it is written and conceived, is more like developing a tailor-made method for quantification of fungi in Daqu, and not very useful for widely fungal communities. This is a serious shortcoming of work.
In general, the paper lacks in the introduction and in the discussion a serious review of the need to develop such a method. The need to develop a new method from the point of view of currently developed methods is well explained, but it is not enough for publication.
If consider the paper as a new method for quantification of fungi species only in Daqu, then there is no description of the current methods used and of course a description of the problem in terms of why it is necessary to quantify fungal species. Because, as far as we know, Daqu is a commercially available product and its production obviously does not depend on the exact identification of the fungal species that participate in its production. Therefore, my suggestion is to present the results in a completely new light with clear guidelines on how the developed method can be used for multiple purposes with examples. Authors only at the end of the conclusion actually state the key sentence that should shape the purpose of the whole research. As far as I can see, the authors have more clearly emphasized the purpose and importance of quantification of species in their previous work.
Further, from all presented results and methods it is not clear how authors identified core fungi? How they known all these species is existing in Daqu? Is it known form previous research or from manufactory recipes? Or authors used named ITS for identification? This must certainly be added to the text or an identification shown if it is now done. And was the identification of all species present possible using only ITS? Internal standard primers were chosen according to ITS length, OK (good idea). But, authors further discus and present results according to species. I do not see clear correlation between ITS length and species names, please state it in text.
A paper written in this way does not fit well enough into the chosen journal. Data on the fermentation process during which Daqu is produced are missing.
My suggestion is that the work be rewritten either in the direction of a method that is useful for the specification of fungal communities or for the specification of fungi present in the production of Daqu. If the authors choose the second variant, then the paper could be reconsidered for publication in Fermentation, but with a serious consideration of the influence of the fungal community in the production process. In terms of changes in the quality of the final product or similar. Case studies examined here need to be better discussed in terms of significance and differences between the temperatures used in fermentation. It is not clear from the text why there are fermentations at different temperatures or what is their impact on fungi or the final product. Discussion sector is unfairly limited to the details of the method only.
Additional, lines 48-50 please rewrite to be shortly and concise
Table 2. identification of fungi is missed, what mean source column if it is already the same for everyone? description of the abbreviation and b are missing.
etc...
minor suggestion would be usefull only in revised form if it will available to me.
Author Response
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Author Response.pdf
Reviewer 2 Report
The authors developed a high-throughput method for quantification of fungal species in fermented products. Using this method, the authors tried to quantify the core fungal species in Daqu used as starter of Chinese liquors.
The theme of this manuscript is interesting, and fit to the Journal “Fermentation”. However, some results and experimental procedures are not clearly described in this manuscript, making it difficult to understand.
[Major points]
Figure 4 (standard curve).
In section 2.4, the authors indicated that the standard carve was generated using 10-fold serial dilution of “ISP2”. However, details of the standard curves for 16 fungal species were not described. What kinds of experimental data were used in obtaining the standard carves of 16 fungal species (qPCR? or Illumina MiSeq sequencing?). I think the standard curves are one of the key points of this study. Thus, the detail protocol for obtaining the standard curves of 16 fungal species are necessary to describe clearly.
Line 162-165 (protocol of sequence analysis)
What does “the high-throughputs” mean?
Did authors amplify fungal ITS regions in the samples (i.e. Daqu with/without ISP) before sequence analysis using Illumina MiSeq sequencer?
It is also very important for this study that how did authors analyze ITS sequences and the spikes in the samples.
Section 2.3.(Mature Daqu samples and experimental setup)
Kinds of ingredients (and origin of the ingredients), sizes and shapes (blocks? cubes? or mushs?), and incubation conditions (temperature, relative humidity, air flow etc.) of Daqu samples were not indicated in this manuscript.
Temperature and humidity should be described numerically.
“High”, and “middle-high” are not sufficient for publishing in scientific journals.
Order of Tables.
The tables to be ordered from smallest to largest.
In this manuscript, Table 2 was indicated before Table 1. It may cause confusion of the reader of the Journal.
Section 3.2, line 231-242
The second choice (1.0 x 107 copies/mL) was included in the first concentration range. Thus, the logic of this text is unreasonable (the second choice is not “other” suitable concentration). This may obstacle to understanding of the readers.
I recommend to revise the text in section 3.2.
(All the results indicated in Figure 2 were used in determining the suitable concentration of ISP2. These tree data may describe as one group, “the first choice” and “the second choice” may not be necessary to divide.)
[Minor points]
Line 88
ISP and ISF -> ISP (internal standard plasmids) and ISF (internal standard fragments)
Although these abbreviations were explained in the section of Results (line 209-211),
I recommend to add these descriptions in the section of Materials and Methods too.
Line 122
What does “Qufang” means?
Brief explanations of “Qufang” might be required.
Reference No.28
Please translate the author name(s), title of the paper, and Journal name in English.
Section 2.1
In general, pET vectors including pET-28a are regarded as strong protein expression vectors because pET vectors have strong T7 promoter. However, protein overproduction has not been done in this study.
Why did the authors use pET-28a in this study?
Are there some reasons for using pET-28a?
[Other comments]
Can the high-throughput method detect quantity and species of fungi at the same time?
Do bacteria contribute to fermentation (or maturation) of Daqu?
Where the fungal species in Daqe come from?
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript by Du et al. attempts to describe a method that employs NGS sequencing of ITS amplicons, coupled with normalization by spiked-in standard sequences, to perform absolute quantification of different fungal taxa present in complex communities. Authors built the rationale for the present work from a previously published manuscript of their own authorship, in which the absolute quantification of bacterial communities has been performed with the aid of indigenous internal standards [Du, R., Wu, Q., & Xu, Y. (2020) Applied and environmental microbiology, 86(12), e00456-20].
While the development of new approaches to obtain absolute quantification of microorganisms from NGS data is a worthy contribution the study of microbiomes, I fear that the current manuscript is EXTREMELY confusing in explaining the overall approach proposed by the authors. In fact, the Discussion presented by the authors does a better job in describing their rationale than all previous parts of the manuscript.
In their previous manuscript (Du et al., 2020), authors employed a straightforward approach, based on the constant ratios of Relative Quantification/Absolute Quantification to obtain the semi-quantitative estimates of different bacterial taxa. To accomplish that, authors relied on NGS to obtain Relative Quantifications and on qPCR of indigenous internal standards to obtain Absolute Quantifications from some control taxa. Unfortunately, I could not understand how the Absolute Quantifications were obtained in the current manuscript (NGS? qPCR? – in the latter case, which primers were employed, to discriminate spiked-in ISFs from true ITS amplicons?).
Moreover, authors introduced a series of new variables to estimate Absolute Quantification of fungal taxa, but did not explain them properly. What are the roles of E and M in equation (2), described between lines 182-183 [shouldn´t M be described as mass (grams), instead of weight?].
In summary, I recognize that the manuscript has the potential to describe a useful approach to obtain absolute taxa quantification from mycobiome NGS data, but cannot provide a more detailed evaluation of the work, in its current status.
Thus, I suggest that authors conduct a THOROUGH revision on their manuscript, providing a more careful description of their rationale and data. Particular attention should be paid to figure legends, which provide extremely inaccurate experimental descriptions.
Author Response
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Round 2
Reviewer 1 Report
Response 1:
please give short explanation in introduction section, not only in response
Response 2:
please provide one-two sentence in disscusion section, too
Response 3:
unnecessary sarcasm. Lines 375-379, p 9 correspond more to the discussion section than to the results. if you were to move to the discussion section you would respond better to my remark, for example. I sure that you can add some similar sentences in discusion.
lines 523-533 p15 - same as above
Disscusion sector is still insuficinece in some descriptions, I thing that some sentences from results coul be rewritted for disscusion.
p18, lines 650-657 I do not understand why authors deleted?
Author Response
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Reviewer 2 Report
Although many revision histories are indicated in the revised manuscript, most of the texts in the manuscript have not been changed from previous version. Thus, the manuscript is not properly revised. Unfortunately, this manuscript is not sufficient for publishing in fermentation.Author Response
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Author Response File: Author Response.pdf
