A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin

Takuhei Shiozaki; Fumihiro Itoh; Yuu Hirose; Jonaotaro Onodera; Akira Kuwata; Naomi Harada

doi:10.1371/journal.pone.0245936

Peer Review History

Original SubmissionNovember 4, 2020
3 Dec 2020 Decision Letter - Arga Chandrashekar Anil, Editor PONE-D-20-34778 A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin PLOS ONE Dear Dr. Shiozaki, 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. The two reviews obtained indicate the contribution useful and have made comments that need your consideration and appropriate incorporation. Please submit your revised manuscript by Jan 17 2021 11:59PM. 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. 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Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 ****** 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 ****** 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: Manuscript ID: PONE-D-20-34778 Reviewer’s comments In this manuscript by Shiozaki et al entitled “A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin”, the authors developed a method for extracting DNA from archived formalin-preserved plankton samples to determine their community structure by a DNA metabarcoding approach and indicated its use to describe the original community structure of plankton stored in formalin for years. This approach is useful for examining the long-term variation of planktons by metabarcoding analysis of 18S rRNA gene community structure. The manuscript is relatively well written but needs improvement as there are many grammatical errors throughout the manuscript. In my opinion, this paper has a good potential to be published in the journal and can be accepted after minor revision. My concerns and suggestions are as follows: The introduction section is well written. In the methods section, my main concern is the lack of description of some key aspects of the experimental design such as the number of replicates analysed, description of samples etc. that prevent assessment of the statistical soundness of the results. The methods need to be made clearer. In view of this, a schematic on the experimental design emphasizing on the sampling and different extraction protocols will be useful. Also, since the focus of this paper is on metabarcoding analysis of 18S rRNA gene community structure it would be useful to provide details on composition of the eukaryotic community in the main manuscript instead of including in the supplementary file 2. The rational for using sample S, which was not formalin fixed as a positive control and not sample E (time zero) which was also not fixed with formalin need to be provided. In the case of results section, especially for long-term preservation in formalin, it will be helpful if the authors refer to the samples with respect to age (plankton samples that were preserved for 16 and 23 years and the sediment trap samples stored for 17 years) or type (plankton/sediment) instead of codes such as ABCD as it is confusing. Also, when comparing the results of the microscopy and DNA analysis, the interpretation’s provided for the discrepancies has to be realistic. Besides, the sub-title Time-variation in an entire plankton community after formalin fixation may be replaced with “Influence of time on the entire plankton community after formalin fixation”. Reviewer #2: The paper is nicely written and easy to follow. The authors test how formalin fixation affects the DNA content of plankton samples that have been stored for many years. They test nine different DNA extraction methods on samples stored in formalin for up to 23 years. They also provide a comparison of changes in plankton community for samples collected at the same time and place where the DNA was extracted immediately for one sample and stored for 0.5 and 1.5 years for two additional samples. They examine the success of DNA extraction using real-time PCR, metabarcoding of the V7-V8 region of the 18S rRNA gene as well as traditional cell counting of diatoms. Their findings can be useful for other researchers who are working with samples preserved in formalin. Some minor comment: L21: “tuned to” should be “turned to”. L34: “have been examined to evaluate”, change to “have evaluated”. L35: “planktons” should be “plankton diversity” L242: I can’t see “PC” in figure 2. L299 (and elsewhere): The authors use SV as an abbreviation for the sequence variants from Dada2. However, the majority of publications use ASV, short for amplicon sequence variation. ASV is also the term preferred by the authors of Dada2. Figure 2: There is no title in the small boxed legend in the figure (numbered 1-9 + std) . I guess it refers to lysis solution in table 2. The figure would be easier to read if the authors could fit a small description of the lysis buffer next to the number. (E.g. “1: BNB, pH 11” for the first “2: PB, pH 8.5” for the second, and so on). Figure 3: I very much prefer supplemental figure S2 to the pie charts used in this figure. I suggest that authors make figure S2 slimmer by removing the full taxonomic assignment for the SVs (which should be renamed ASVs), and keep only the final part of the name (i.e. Phaeocystis poechetii, Eunicida sp., Calocalanus pavo, etc). The color scheme can be kept to separate between different supergroups. For the colors I would also differentiate between Metazoa and Fungi. Finally, I think it makes more sense to put the haptophytes underneath the fungi, together with SAR. Then S2 could probably be the main Figure 3.. Figure 5. The names can be shortened as described for the previous figure. Also I the ASVs it could be ordered more logically from top to bottom: Metazoa, Choanoflagellates, Fungi, Apusomonadidae, Picozoa, Archaeplastida, Telonema, Rhizaria, Alveolata, Stramenopiles (see Burki et al. 2019, The New Tree of Eukaryotes, and Adl et al 2019 Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes) ****** 6. 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Revision 1
22 Dec 2020 Author Response Reviewer #1: Manuscript ID: PONE-D-20-34778 Reviewer’s comments In this manuscript by Shiozaki et al entitled “A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin”, the authors developed a method for extracting DNA from archived formalin-preserved plankton samples to determine their community structure by a DNA metabarcoding approach and indicated its use to describe the original community structure of plankton stored in formalin for years. This approach is useful for examining the long-term variation of planktons by metabarcoding analysis of 18S rRNA gene community structure. The manuscript is relatively well written but needs improvement as there are many grammatical errors throughout the manuscript. In my opinion, this paper has a good potential to be published in the journal and can be accepted after minor revision. My concerns and suggestions are as follows: The introduction section is well written. In the methods section, my main concern is the lack of description of some key aspects of the experimental design such as the number of replicates analysed, description of samples etc. that prevent assessment of the statistical soundness of the results. The methods need to be made clearer. In view of this, a schematic on the experimental design emphasizing on the sampling and different extraction protocols will be useful. We have corrected grammatical errors throughout the manuscript. We have added the number of samples used in the experiments at L88, 104-107, and 110. The only difference in the extraction protocol is the lysis solution used. Thus, we have added which lysis solution was used for each sample in Table 1. Also, since the focus of this paper is on metabarcoding analysis of 18S rRNA gene community structure it would be useful to provide details on composition of the eukaryotic community in the main manuscript instead of including in the supplementary file 2. We have moved Figure S2 to the main text. The rational for using sample S, which was not formalin fixed as a positive control and not sample E (time zero) which was also not fixed with formalin need to be provided. This is a practical reason. DNA extraction of sample E had not yet been performed when the fluorescent PCR experiment was conducted. In the case of results section, especially for long-term preservation in formalin, it will be helpful if the authors refer to the samples with respect to age (plankton samples that were preserved for 16 and 23 years and the sediment trap samples stored for 17 years) or type (plankton/sediment) instead of codes such as ABCD as it is confusing. We have added preservation term and sample type in the result section (L231, 272, 275, 278, 295, and 316). Also, when comparing the results of the microscopy and DNA analysis, the interpretation’s provided for the discrepancies has to be realistic. The differences between the two methods are Coscinodiscus, Porosira, and Neodenticula. As mentioned in the manuscript, this difference can be explained by problems with the DNA database and misidentification by microscopy. Besides, the sub-title Time-variation in an entire plankton community after formalin fixation may be replaced with “Influence of time on the entire plankton community after formalin fixation”. We have changed as suggested. Reviewer #2: The paper is nicely written and easy to follow. The authors test how formalin fixation affects the DNA content of plankton samples that have been stored for many years. They test nine different DNA extraction methods on samples stored in formalin for up to 23 years. They also provide a comparison of changes in plankton community for samples collected at the same time and place where the DNA was extracted immediately for one sample and stored for 0.5 and 1.5 years for two additional samples. They examine the success of DNA extraction using real-time PCR, metabarcoding of the V7-V8 region of the 18S rRNA gene as well as traditional cell counting of diatoms. Their findings can be useful for other researchers who are working with samples preserved in formalin. We appreciate for your constructive comments. We have corrected the points you raised as follows. Some minor comment: L21: “tuned to” should be “turned to”. Changes as suggested. L34: “have been examined to evaluate”, change to “have evaluated”. Changed as suggested. L35: “planktons” should be “plankton diversity” Changed as suggested. L242: I can’t see “PC” in figure 2. We have reworded “positive control” in the revised figure. L299 (and elsewhere): The authors use SV as an abbreviation for the sequence variants from Dada2. However, the majority of publications use ASV, short for amplicon sequence variation. ASV is also the term preferred by the authors of Dada2. We have changed the wording from SV to ASV throughout the manuscript. Figure 2: There is no title in the small boxed legend in the figure (numbered 1-9 + std) . I guess it refers to lysis solution in table 2. The figure would be easier to read if the authors could fit a small description of the lysis buffer next to the number. (E.g. “1: BNB, pH 11” for the first “2: PB, pH 8.5” for the second, and so on). We have added the information of the lysis buffer in the legend. Figure 3: I very much prefer supplemental figure S2 to the pie charts used in this figure. I suggest that authors make figure S2 slimmer by removing the full taxonomic assignment for the SVs (which should be renamed ASVs), and keep only the final part of the name (i.e. Phaeocystis poechetii, Eunicida sp., Calocalanus pavo, etc). The color scheme can be kept to separate between different supergroups. For the colors I would also differentiate between Metazoa and Fungi. Finally, I think it makes more sense to put the haptophytes underneath the fungi, together with SAR. Then S2 could probably be the main Figure 3. We have moved Figure S2 to the main text, made figure slimmer, and changed the order as suggested. Figure 5. The names can be shortened as described for the previous figure. Also I the ASVs it could be ordered more logically from top to bottom: Metazoa, Choanoflagellates, Fungi, Apusomonadidae, Picozoa, Archaeplastida, Telonema, Rhizaria, Alveolata, Stramenopiles (see Burki et al. 2019, The New Tree of Eukaryotes, and Adl et al 2019 Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes) We have changed the order as suggested. https://doi.org/10.1371/journal.pone.0245936.r002
11 Jan 2021 Decision Letter - Arga Chandrashekar Anil, Editor A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin PONE-D-20-34778R1 Dear Dr. Shiozaki, 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, Arga Chandrashekar Anil, Ph. D., D. Agr., Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0245936.r003
Formally Accepted
26 Jan 2021 Acceptance Letter - Arga Chandrashekar Anil, Editor PONE-D-20-34778R1 A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin Dear Dr. Shiozaki: 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 Professor Arga Chandrashekar Anil Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0245936.r004

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