The effect of soil sample size, for practical DNA extraction, on soil microbial diversity in different taxonomic ranks

Hiroki Morita; Satoshi Akao

doi:10.1371/journal.pone.0260121

Peer Review History

Original SubmissionJune 24, 2021
4 Aug 2021 Decision Letter - Ashwani Kumar, Editor PONE-D-21-20731 The effect of soil sample size, for practical DNA extraction, on soil microbial diversity in different taxonomic ranks PLOS ONE Dear Dr. %Satoshi%, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ACADEMIC EDITOR: Ashwani Kumar Please submit your revised manuscript by %20-08-2021%. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Ashwani Kumar Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in your Competing Interests section: [NO]. Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 4. We note that S-Figures 1 and 2 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: a) You may seek permission from the original copyright holder of Figures S-Figure 1 and S-Figure 2 to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” b) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: your work "the effect of soil sample size, for practical DNA extraction, on soil microbial diversity in different taxonomic ranks" is very interesting and useful for cauterising soil and understanding the nature of microorganisms presentation. Reviewer #2: The manuscript entitled ‘The effect of soil sample size, for practical DNA extraction, on soil microbial diversity in different taxonomic ranks' tried to determine the optimal soil sample size in regard for microbial community structure. They focused on the relationship between soil amount and DNA extraction container volume and the alteration in microbial composition at different taxonomic ranks (order, class, and phylum). Three soil sample size were tested, and they concluded that microbial diversity and diversity assessment were stable on 0.2 and 1-g samples. It is interesting to found the effect of sample size to microbial diversity. Thus, I think it is valuable to publish the paper, which would provide technical insight of sampling size. However, I would like to propose a small suggestion to the author to adjustment the conclusion. It would be easier for the reader if the conclusion is brief and concise, and which sample size practice is the best. Reviewer #3: General comments This manuscript reports on the optimal soil sample size for microbial community structure analysis. Besides, this study also focused on the relationship between soil amount and DNA extraction container volume and the alteration in the microbial composition at different taxonomic ranks. The results suggest that microbial community structure analysis is influenced by the taxonomic rank applied. However, I have some concerns about some of the methodology used in this study. For example, the sample sizes used and also the number of genes used in the microbial composition analysis. These indirectly impacts the general results and findings of this study. Moreover, the authors need to clarify the novelty of their study as the same study has been shown in other research, Penton et al., 2016, even though it has been cited in the study. The authors should make this clear in the introduction and make a more comprehensive conclusion. Hence, I have to reject this manuscript because it does not meet the PLoS ONE criteria However, these studies still have an impact of results in its own way * Consider carefully proofreading – there are several grammatical errors and also replicate of a sentence. ******** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0260121.r001
Revision 1
8 Sep 2021 Author Response Reviewer #1: your work "the effect of soil sample size, for practical DNA extraction, on soil microbial diversity in different taxonomic ranks" is very interesting and useful for cauterising soil and understanding the nature of microorganisms presentation. Author: Thank you very much for your encouraging words. Reviewer #2-1: The manuscript entitled ‘The effect of soil sample size, for practical DNA extraction, on soil microbial diversity in different taxonomic ranks' tried to determine the optimal soil sample size in regard for microbial community structure. They focused on the relationship between soil amount and DNA extraction container volume and the alteration in microbial composition at different taxonomic ranks (order, class, and phylum). Three soil sample size were tested, and they concluded that microbial diversity and diversity assessment were stable on 0.2 and 1-g samples. It is interesting to found the effect of sample size to microbial diversity. Thus, I think it is valuable to publish the paper, which would provide technical insight of sampling size. Author:We appreciate the reviewer’s high evaluation. Reviewer #2-2: However, I would like to propose a small suggestion to the author to adjustment the conclusion. It would be easier for the reader if the conclusion is brief and concise, and which sample size practice is the best. Author: Thank you very much for the useful suggestion. We have concluded that the 0.2-g soil sample level would be preferable due to its higher detection of bacteria with a high tolerance of shearing force and its economic efficiency [L. 31 and 402]. Also, we made the Abstract and the Conclusion briefer and more concise [L. 17, 27, 30, 393, and 401]. Reviewer #3-1: General comments This manuscript reports on the optimal soil sample size for microbial community structure analysis. Besides, this study also focused on the relationship between soil amount and DNA extraction container volume and the alteration in the microbial composition at different taxonomic ranks. The results suggest that microbial community structure analysis is influenced by the taxonomic rank applied. However, I have some concerns about some of the methodology used in this study. For example, the sample sizes used and also the number of genes used in the microbial composition analysis. These indirectly impacts the general results and findings of this study. Author: Thank you very much for your meaningful comments. As the reviewer mentioned, the soil sample size might affect the microbial composition analysis. Also, we have focused on the relationship between the soil sample size and the DNA extraction conditions (e.g., the volume of the container). Regarding the study using amplicon sequence data, Penton et al. and Song et al. have studied the soil sample size effect. However, However, Penton et al. evaluated the effect of the soil sample sizes (1, 5, and 10 g) using the DNA extraction kit for 10 g of soil, with the result that a larger soil sample size would be preferable. In this respect, each DNA extraction kit would have an optimal soil volume, and the smaller soil sample size in the study might deviate from the optimal volume. In other words, the relationship between the soil volume and the container volume for DNA extraction would be important, as well as the soil sample size effect, and we have investigated the points. Song et al. also elucidated the DNA extraction efficiency using the different soil sample sizes (0.25 g and 10 g) with each optimal-size DNA extraction kit, respectively. There was no significant difference in the OUT richness between the two soil sample sizes, and this result would support our idea, meaning the relationship between the soil volume and the container volume is also essential. Therefore, we added the report by Song et al. in the reference list [L. 71]. We would have liked to carry out further soil sample size cases. However, the horizontal shaking adapter for 50 mL tube volume for the vortex mixer can only equip two tubes. It is inconvenient for practical use because we usually treat many samples at one time. Therefore, we have tried a vertical, not a horizontal shaker, which can equip 6 tubes at once, to extract DNA for practical use. We would think that the sample size indicated by Reviewer#3 means the soil sample size. However, generally, sample size means repetition of one sample. Therefore, to distinguish soil sample volume from the so-called sample size, we added the word “soil” prior to sample size, meaning soil sample volume. Also, we apologize for the confusion. Relating to the so-called sample size, we also added the reason for our repetition number of one sample (n = 5) by referring to Song et al. (2014) and Vestergaard et al. (2017) [L.50 and 117]. The number of genes also would affect microbial composition analyses. We think the reviewer’s indication has two meanings: one is the volume of sequence reads, and the other is the equality of the sequence reads by rarefaction. For the former points, 30,000-40,000 reads were obtained through the experimental protocols in our study. Therefore, the sequence reads would be enough to evaluate microbial composition analysis (Schöler et al., 2017). Also, we rewrote the sequence reads of Penton et al. from its total reads (over 5,000,000 reads) to the reads of each sample (over 56,000 reads) for the comparison of our sequence reads [L. 69]. For the latter points, we used the same soil, not the different soil. So, the number of sequence reads was one of the consequences of comparing the different soil sample sizes for DNA extraction. Therefore, we did not rarefy the read number and conducted the microbial community analysis using the obtained read number. We mentioned the point in the revised manuscript [L. 193]. By the way, the average number of sequence reads in each soil sample size would be confirmed to be almost the same (no significant difference) by applying ANOVA [L. 161]. Reviewer #3-2: Moreover, the authors need to clarify the novelty of their study as the same study has been shown in other research, Penton et al., 2016, even though it has been cited in the study. The authors should make this clear in the introduction and make a more comprehensive conclusion. Author: Thank you for your advice. Our main discussion points are as follows; 1) effects of the target taxonomic rank and annotated taxa in microbial diversity analysis and 2) the relationship between soil volume and tube volume for DNA extraction. Firstly, we summarized the section of our motivations in an orderly way (firstly, secondly, finally) in the Introduction section [L. 76, 88, and 98]. Then, we noticed that the explanation of the former discussion point, especially using annotated taxa for microbial diversity analysis, had not been adequate in the original manuscript, so we described the point in more detail [L. 73 and 108]. We added concerns about using OTUs not annotated taxa for microbial diversity analysis [L. 76]. Also, we referred to the report by Song et al. (2015) to support our discussion [L. 71, 95]. By the way, we found our misunderstanding about the target taxonomic rank in the study by Penten et al. (2016). They did not determine a specific taxonomic rank. We apologize and corrected the point [L. 76]. We also discussed the importance of setting target taxonomic rank and using annotated taxa in microbial diversity analysis in the Results and Discussion section. We added some ideas for the transition of the Sørensen-Dice index with lowering the taxonomic rank relating to the detection of minor taxa. In addition, we wrote the possible reason for the different conclusions about detecting minor taxa between ours and Penton et al.’s. [L.339 and 348]. We also added the reference that discussed soil sample size for the detection of minor taxa [L. 355]. We re-evaluated our results for a more straightforward conclusion and concluded that a 0.2-g soil sample size would be better than a 1-g soil sample size because the soil sample size could detect taxa with higher physical shear tolerance and was carried out at a lower cost. We rewrote the choice of the two soil sample sizes in the Results and Discussion section (Determination of Sample Size) [L. 364] and the Conclusions section [L. 402]. In the Determination of Sample Size section, we summarized the advantages of each soil sample size (0.2 and 1 g) [L. 363]. We added the following contents to explain the points mentioned (underline shows the added sentences and phrases). In addition, we cleared some information in the Introduction that did not relate to the main discussing points to avoid redundancy [L. 43]. Also, we summarized the Conclusion to make it more concise [L. 393]. Reviewer #3-3: Hence, I have to reject this manuscript because it does not meet the PLoS ONE criteria However, these studies still have an impact of results in its own way * Consider carefully proofreading – there are several grammatical errors and also replicate of a sentence. Author: Thank you for your advice. We have again discussed our novelty and the differences from previous reports and revised the manuscript as we mentioned above. We have sincerely read the revised manuscript and used the English proofreading service again. Attachments Attachment Submitted filename: 210904_Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0260121.r002
3 Nov 2021 Decision Letter - Ashwani Kumar, Editor The effect of soil sample size, for practical DNA extraction, on soil microbial diversity in different taxonomic ranks PONE-D-21-20731R1 Dear Dr. Akao, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ashwani Kumar Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0260121.r003
Formally Accepted
8 Nov 2021 Acceptance Letter - Ashwani Kumar, Editor PONE-D-21-20731R1 The effect of soil sample size, for practical DNA extraction, on soil microbial diversity in different taxonomic ranks Dear Dr. Akao: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ashwani Kumar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260121.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .