Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide

Takahiro Yamagishi; Haruyo Yamaguchi; Shigekatsu Suzuki; Mayumi Yoshikawa; Ian Jameson; Maike Lorenz; David R. Nobles; Christine Campbell; Masanori Seki; Masanobu Kawachi; Hiroshi Yamamoto

doi:10.1371/journal.pone.0241889

Peer Review History

Original SubmissionAugust 20, 2020
25 Sep 2020 Decision Letter - Yi Cao, Editor PONE-D-20-26198 Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide PLOS ONE Dear Dr. Yamagishi, 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. Please submit your revised manuscript by Nov 09 2020 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. 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 We look forward to receiving your revised manuscript. Kind regards, Yi Cao Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 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. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 'This research is partially supported by the National BioResource Project (NBRP) from Japan Agency for Medical Research and Development (AMED). We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. a. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 'The author(s) received no specific funding for this work.' Please ensure your amended statement declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now If partially funded, please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. b. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary). [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: No ****** 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: Thank you for the opportunity to read and review this manuscript. The authors have performed a comparative study looking into the genetic variation of 9 culture collection strains of Raphidocelis subcapitata, all of which were derived from the same isolated strain NIVA-CHL1. Variants (SNPs and indels) were called in each strain relative to the reference genome and were assessed for their potential impacts on protein function. Two strains (ATCC 22662-2 and CCAP 278/4) contained mutations in an ion channel protein and growth inhibition tests with NaCl indicated that these mutations might influence osmotic regulation. All 9 strains additionally showed vast differences in toxicant sensitivity. The authors suggest that these differences may be due to acquired mutations from long-term sub-culturing under different conditions. The authors present a very interesting study that has clear implications for those studying microalgal strains housed in culture collections. It should not be surprising that genetic variation gets acquired during long-term culturing under different conditions. The results presented here offer convincing evidence that laboratory cultures of the same strain act differently. I would also argue that these points about random genetic mutation should be taken into consideration for all labs that work with model organisms. The authors point out the important need to standardize the maintenance of model algal strains and that cryopreservation offers some benefit. Overall, I found this manuscript to be well-written and, given the stated goals of the study, the results are convincing and presented well. The methods were appropriate. The figures were useful and designed well. Many of my suggestions that I detail below are to clarify or provide additional details about how some of the methods were performed. Suggestions: Line 140: Provide information about whether the libraries were barcoded (single or dual indices) and any pooling details. Line 143: What was the length of the sequenced reads produced from the HiSeq X Ten and MiSeq systems? Were they single- or paired-end reads? Line 150: More details should be included about variant calling in GATK. For instance, was the GATK Best Practices followed (https://www.broadinstitute.org/partnerships/education/broade/best-practices-variant-calling-gatk-1), or a modified pipeline? Was indel realignment performed? Was base quality score recalibration performed? Were the initial variants filtered for quality or depth? Line 254: I would add here in parentheses what the bootstrap values were for these relationships that you are describing. Line 328: “sensitivity of chemical substances” to “sensitivity to chemical substances.” Line 364: “in genes of 9-15 genes” to “in 9-15 genes” Reviewer #2: Generally, Raphidocelis subcapitata was one of the most frequently used species for algal growth inhibition tests. Therefore, genetic characteristics of R. subcapitata was very crucial for its functional applications. This manuscript compared the genome sequences among NIVA-CHL1 from 9 microalgal culture collections to evaluate the presence of mutations. The results was very meaningful for subsequent applications by different mutations. However, some issues need to be addressed as the following point to point: 1) In Introduction Section, the author should describe the significance of this study in investigating mutants. 2) In Materials and Methods Section (line 131), the strains were propagated under continuous light at 23°C in OECD medium. The illumination intensity and the compositions in OECD medium should be shown. 3) In line 138, 10-mL should be changed into 10 mL, please unify this similar situation in the whole manuscript. 4) In line 186, it would be better to note the source of the species by references in Table 1. 5) In the Growth inhibition tests, two chemicals were used: 3,5-DCP (Lot: SDK3769, > 98.0%) and sodium chloride (NaCl) (Lot: 7935, >99.5%), however, the concentrations of the two chemicals in the medium were not mentioned. Please described in detail. 6) Please carefully check grammatical and typing errors in the whole manuscript and please provide a higher resolution data graphs. In sum, I recommend publication of the paper in PLOS ONE, provided the authors comply with my comments above. ****** 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 [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.0241889.r001
Revision 1
15 Oct 2020 Author Response Dear editors, Thank you very much for your efforts in editing our submission. We revised the manuscript considering the valuable comments by you and the reviewers. Regarding one major point of the suggestions from Reviewer 1 on providing additional information how the genome sequence and analyses were performed, we revised the manuscript adding more information about this. Also, we sincerely apologize for the grammatical and careless typographical errors in original submission. We have made a thorough check of this revised version, and hope that no errors remain. In addition, we responded to all of Journal Requirements on style, funding information in Acknowledgements, and so on. Please see the following, indicating our responses. Review Comments to the Author Reviewer #1: Thank you for the opportunity to read and review this manuscript. The authors have performed a comparative study looking into the genetic variation of 9 culture collection strains of Raphidocelis subcapitata, all of which were derived from the same isolated strain NIVA-CHL1. Variants (SNPs and indels) were called in each strain relative to the reference genome and were assessed for their potential impacts on protein function. Two strains (ATCC 22662-2 and CCAP 278/4) contained mutations in an ion channel protein and growth inhibition tests with NaCl indicated that these mutations might influence osmotic regulation. All 9 strains additionally showed vast differences in toxicant sensitivity. The authors suggest that these differences may be due to acquired mutations from long-term sub-culturing under different conditions. The authors present a very interesting study that has clear implications for those studying microalgal strains housed in culture collections. It should not be surprising that genetic variation gets acquired during long-term culturing under different conditions. The results presented here offer convincing evidence that laboratory cultures of the same strain act differently. I would also argue that these points about random genetic mutation should be taken into consideration for all labs that work with model organisms. The authors point out the important need to standardize the maintenance of model algal strains and that cryopreservation offers some benefit. Overall, I found this manuscript to be well-written and, given the stated goals of the study, the results are convincing and presented well. The methods were appropriate. The figures were useful and designed well. Many of my suggestions that I detail below are to clarify or provide additional details about how some of the methods were performed. Suggestions: Line 140: Provide information about whether the libraries were barcoded (single or dual indices) and any pooling details. Considering this comment, we revised as follows: “Genomic libraries of paired-end reads were constructed and barcoded using an NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, USA) and……”(p.7, line 144-146). Line 143: What was the length of the sequenced reads produced from the HiSeq X Ten and MiSeq systems? Were they single- or paired-end reads? Considering this comment, we revised as follows: “Genomic libraries of paired-end reads were constructed and barcoded using an NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, USA) and NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (New England Biolabs). The library concentrations and size distributions were determined with NEBNext Library Quant Kit for Illumina (New England Biolabs) and Agilent 2200 Tape Station (Agilent Technologies, USA). Equinanomolar concentrations from each library were pooled. Next-generation sequencing was performed on a HiSeq X Ten system instrument as 150 bp paired-end reads by Novogene Corporation (Beijing, China) or on a MiSeq system instrument as 300 bp paired-end reads.”(p. 7-8, line 144-153). Line 150: More details should be included about variant calling in GATK. For instance, was the GATK Best Practices followed (https://www.broadinstitute.org/partnerships/education/broade/best-practices-variant-calling-gatk-1), or a modified pipeline? Was indel realignment performed? Was base quality score recalibration performed? Were the initial variants filtered for quality or depth? Considering this comment, we revised as follows: “The variant calling was performed according to the GATK best practices (http://software.broadinstitute.org/gatk/best-practices). Since there is no known variant data, base quality score recalibration was not performed. SNPs and indels were classified in snpEff ver.4.2 [7](http://snpeff.sourceforge.net/SnpEff_manual.html).”(p. 8, line 161-164). Line 254: I would add here in parentheses what the bootstrap values were for these relationships that you are describing. We added the bootstrap value (p. 15, line 270). Line 328: “sensitivity of chemical substances” to “sensitivity to chemical substances.” We corrected this (p. 19, line 343). Line 364: “in genes of 9-15 genes” to “in 9-15 genes” We corrected this (p. 20, line 386). Reviewer #2: Generally, Raphidocelis subcapitata was one of the most frequently used species for algal growth inhibition tests. Therefore, genetic characteristics of R. subcapitata was very crucial for its functional applications. This manuscript compared the genome sequences among NIVA-CHL1 from 9 microalgal culture collections to evaluate the presence of mutations. The results was very meaningful for subsequent applications by different mutations. However, some issues need to be addressed as the following point to point: 1) In Introduction Section, the author should describe the significance of this study in investigating mutants. Considering this comment, we added the following sentence: “In addition, it is critical issue for the standard strain whether sensitivities to toxicants vary among the strains maintained at culture collections worldwide.”(p. 6, line 110-111) 2) In Materials and Methods Section (line 131), the strains were propagated under continuous light at 23°C in OECD medium. The illumination intensity and the compositions in OECD medium should be shown. Considering this comment, we revised as follows: “The strains were propagated under continuous light (60–80 μmol·m−2·s−1) at 23°C in OECD medium [1].”(p. 7, line 134) 3) In line 138, 10-mL should be changed into 10 mL, please unify this similar situation in the whole manuscript. We corrected this (p. 7, line 142). 4) In line 186, it would be better to note the source of the species by references in Table 1. Considering this comment, we added URL of webpage for the strain information in Table 1 (p. 10). 5) In the Growth inhibition tests, two chemicals were used: 3,5-DCP (Lot: SDK3769, > 98.0%) and sodium chloride (NaCl) (Lot: 7935, >99.5%), however, the concentrations of the two chemicals in the medium were not mentioned. Please described in detail. Considering this comment, we added the following sentence: “(0.25, 0.5, 1.0, 2.0 and 4.0 mg/L in the testing of 3,5-DCP and 111, 333, 1000, 3000 and 9000 mg/L in the testing of NaCl as nominal concentration)” (p. 9, line 182-183) 6) Please carefully check grammatical and typing errors in the whole manuscript and please provide a higher resolution data graphs. We have made a thorough check of this revised version, and provided high resolution figures (TIFF: 300 dpi) to the editorial office together with the revised manuscript. In sum, I recommend publication of the paper in PLOS ONE, provided the authors comply with my comments above. Journal Requirements: 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 We have made a thorough check of this revised version. 2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Regarding the phrase including “data not shown”, we revised as follows: “In our previous study, our alignment of the rbcL regions between NIES-35 and ATCC 22662 showed a mismatch of two base pairs in the rbcL gene [5] (p. 5. Line 100-102)” and “This variability was too large for a test performed in the same laboratory: if a test was performed with different strains in the different laboratories, the difference in sensitivity would be even larger because a result is greatly affected by the differences of handing and test procedure. Thus, we suggest that this difference in sensitivity resulted from genomic mutations (Page 19, line 343-347)”. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 'This research is partially supported by the National BioResource Project (NBRP) from Japan Agency for Medical Research and Development (AMED). We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. I removed any funding-related text from the manuscript, and included our updated statement in our cover letter. 4. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary). We checked this. We hope that these revisions are satisfactory. Sincerely Yours, Takahiro Yamagishi Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0241889.r002
22 Oct 2020 Decision Letter - Yi Cao, Editor Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide PONE-D-20-26198R1 Dear Dr. Yamagishi, 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, Yi Cao Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. 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 ****** 5. 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 ****** 6. 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: Thank you to the authors for considering all comments and making the requested changes to the manuscript. I have read the revised manuscript and have no further comments or suggestions. Reviewer #2: The author has answered and addressed all of my concerns, so I recommend publication in this journal. ****** 7. 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 https://doi.org/10.1371/journal.pone.0241889.r003
Formally Accepted
28 Oct 2020 Acceptance Letter - Yi Cao, Editor PONE-D-20-26198R1 Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide Dear Dr. Yamagishi: 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. Yi Cao Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0241889.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 .