Greater than pH 8: The pH dependence of EDTA as a preservative of high molecular weight DNA in biological samples

Mia L. DeSanctis; Elizabeth A. Soranno; Ella Messner; Ziyu Wang; Elena M. Turner; Rosalia Falco; Hannah J. Appiah-Madson; Daniel L. Distel

doi:10.1371/journal.pone.0280807

Peer Review History

Original SubmissionMay 19, 2022
20 Dec 2022 Decision Letter - Marco Bonizzoni, Editor PONE-D-22-14619Greater than pH 8: the pH dependence of EDTA as a preservative of high molecular weight DNA in biological samplePLOS ONE Dear Dr. Distel, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit and it is very close to fully meeting PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript with minor revisions that address the questions raised during the review process, pertaining to the scope and justification of the work described. Please submit your revised manuscript by Feb 04 2023 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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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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: The manuscript is a great follow-up to a previous paper on DNA preservation using EDTA by the same laboratory (Sharpe et al., 2020, DESS deconstructed: Is EDTA solely responsible for protection of high molecular weight DNA in this common tissue preservative? PLOS ONE, https://doi.org/10.1371/journal.pone.0237356). In that study, the authors demonstrated that EDTA was the primary protector of high-molecular-weight DNA in the common preservative DESS. In this study they evaluate the effectiveness of EDTA as a DNA preservative at various pH’s. The results here are almost as striking and transformative as the previous study, notably so for difficult-to-extract taxa such as Alitta sp. Figure 4 is especially effective in demonstrating the utility of EDTA as a tissue preservative; the results shown in 4a are a bit astonishing. This is an incredibly well-written and edited manuscript; I really have very few suggestions to make the body cleaner and more concise. The rare comments/questions are below. Line 126, 127: Was the fresh tissue from the same individuals as the preserved tissue, which was extracted when the preserved tissues were put into storage? Alternatively, was the fresh tissue from new individuals, extracted with the preserved tissues after 12 months of storage? If the former, please briefly describe how the extracted DNA from fresh tissue was stored for those 12 months. Line 163, 164: Why are the spaces in the sequences for primers LCO1490_t1 and HCO2198_t2 placed where they are? I initially thought it was marking where the primer ended and the M13 tail started, but it does not. I’m not sure if a space is needed in the sequence, but if so, I would think the best place would be between the amplicon primer and the M13 tail. Line 211: You mention taking Absorbance ratios, and include the results in S1 Table, but never mention them at any other time. Were there any significant differences among ratios? Did you run any statistical tests? I find that Absorbance ratios are not very useful and are highly inconsistent in their ability to determine DNA quality, but if they are included, I would possibly mention their results somewhere. Evaluating them with S1 Table is not especially useful. Table 1: I would mention that the p-values are Bonferroni-corrected in the table description. Reviewer #2: dear authors, This is an important study for DNA isolation works but please clearly explain the aims of study in introduction section. What do you want to find from these results? whether these finding can be industrialized or would be just experimentally? Best wishes ****** 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: Yes: Dr. Sakineh Abbasi ******** [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.0280807.r001
Revision 1
6 Jan 2023 Author Response Response to Academic Editors and Reviewers PONE-D-22-14619 Greater than pH 8: the pH dependence of EDTA as a preservative of high molecular weight DNA in biological sample Response to Academic Editor 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 Response: We have renamed the Supporting Information files to match PLOS ONE’s requirements. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Response: We have updated the Funding Information and the Financial Disclosure sections to ensure that they agree. We have also included an additional funder, the Francis Goelet Chartable Lead Trust, which was accidentally omitted from the original submission. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.orgif you have any questions Response: We have included the uncropped gel images in the Supporting Information and have labeled them to indicate the regions of the gels that have been cropped to create Fig 4. All the data used in this investigation has been submitted in the Supporting Information section of our manuscript and so have not been submitted to a public data repository. 4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Response: The complete minimal data set required for this investigation is reported in the Supporting Information section of the manuscript. This includes S1 Table entitled Values for yield (μg), total normalized DNA yield (μg DNA/mg tissue), normalized high molecular weight DNA yield (nY; μg DNA/mg tissue), percent high molecular weight DNA recovered (%R), and COI PCR amplification success for each sample analyzed in this study, S2 Table entitled DNA barcoding, and S1 Fig entitled Raw agarose gel electrophoresis images for qualitative visualization of DNA fragment size distribution after 12 months. 6. 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. Response: We have done so. Response to Reviewer Comments Reviewer #1: The manuscript is a great follow-up to a previous paper on DNA preservation using EDTA by the same laboratory (Sharpe et al., 2020, DESS deconstructed: Is EDTA solely responsible for protection of high molecular weight DNA in this common tissue preservative? PLOS ONE, https://doi.org/10.1371/journal.pone.0237356). In that study, the authors demonstrated that EDTA was the primary protector of high-molecular-weight DNA in the common preservative DESS. In this study they evaluate the effectiveness of EDTA as a DNA preservative at various pH’s. The results here are almost as striking and transformative as the previous study, notably so for difficult-to-extract taxa such as Alitta sp. Figure 4 is especially effective in demonstrating the utility of EDTA as a tissue preservative; the results shown in 4a are a bit astonishing. This is an incredibly well-written and edited manuscript; I really have very few suggestions to make the body cleaner and more concise. The rare comments/questions are below. Line 126, 127: Was the fresh tissue from the same individuals as the preserved tissue, which was extracted when the preserved tissues were put into storage? Alternatively, was the fresh tissue from new individuals, extracted with the preserved tissues after 12 months of storage? If the former, please briefly describe how the extracted DNA from fresh tissue was stored for those 12 months. Response: We have modified the sentences to read as follows: DNA was immediately extracted from the fifth tissue sample of the same individuals without preservation, hereafter referred to as fresh tissue. The DNA from these fresh tissue samples was visualized immediately after extraction by gel electrophoresis and then stored at -80°C for 12 months, at which time DNA from all treatments, including fresh tissue, were analyzed concomitantly (lines 138-143). Line 163, 164: Why are the spaces in the sequences for primers LCO1490_t1 and HCO2198_t2 placed where they are? I initially thought it was marking where the primer ended and the M13 tail started, but it does not. I’m not sure if a space is needed in the sequence, but if so, I would think the best place would be between the amplicon primer and the M13 tail. Response: The extraneous spaces have been removed (lines 183 and 184). Line 211: You mention taking Absorbance ratios, and include the results in S1 Table, but never mention them at any other time. Were there any significant differences among ratios? Did you run any statistical tests? I find that Absorbance ratios are not very useful and are highly inconsistent in their ability to determine DNA quality, but if they are included, I would possibly mention their results somewhere. Evaluating them with S1 Table is not especially useful. Response: We agree with the reviewer’s assessment that the A260/A280 ratios reported in S1 Table are not very useful and that A260/A280 ratios are inconsistent in their ability to determine DNA quality. The A260/A280 ratio is an estimator of DNA purity, which was not examined in this investigation, and we used TapeStation data rather than A260 to estimate DNA concentration. For these reasons, we have removed A260/A280 ratios from the S1 Table and removed mention of them from the text, as suggested by the reviewer (removed from lines 178, 247, and 546). Table 1: I would mention that the p-values are Bonferroni-corrected in the table description. Response: The p values in Table 1 are not Bonferroni-corrected as they apply to single comparisons. However, we applied a Bonferroni correction to all post-hoc tests, e.g., in Figs 2 and 3. Here, the Bonferroni-corrected p value used for post-hoc tests is 0.005 rather than 0.05, reflecting the fact that ten pair-wise comparisons were made. We have corrected the legends of Figs 2 (line 263) and 3 (line 274) as follows: “Within this figure, treatments bearing different lower-case letters are significantly different at the Bonferroni-adjusted p < 0.005” to more accurately reflect the analysis performed. We have also clarified this point in the description of the statistical methods for parametric tests with the following text: “If the data were normal, a one-way repeated measures ANOVA and a Bonferroni-corrected Tukey post-hoc test was performed with the “nlme” package” (lines 237-239). Additionally, in reviewing the statistical analyses we noticed minor error. In one taxon, Faxonius virilis, for %R, we had not applied a Bonferroni correction. We have now done so. As a result, %R for this taxon is not different between EDTA pH 10 and fresh tissue. We have modified the text to reflect this (lines 303, 372-375, 401, and 403). These changes do not affect our conclusion that the performance of EDTA as a preservative of HMW DNA in many tissues improves with increasing pH. Reviewer #2: dear authors, This is an important study for DNA isolation works but please clearly explain the aims of study in introduction section. What do you want to find from these results? whether these finding can be industrialized or would be just experimentally? Response: We have added the following text to the introduction: “Our aim in this investigation is to determine whether the performance of EDTA as a preservative of DNA in tissue can be improved by increasing pH. If so, this simple modification could be of value for research and commercial applications” (lines 95-98). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0280807.r002
10 Jan 2023 Decision Letter - Marco Bonizzoni, Editor Greater than pH 8: the pH dependence of EDTA as a preservative of high molecular weight DNA in biological samples PONE-D-22-14619R1 Dear Dr. Distel, 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, Marco Bonizzoni, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0280807.r003
Formally Accepted
13 Jan 2023 Acceptance Letter - Marco Bonizzoni, Editor PONE-D-22-14619R1 Greater than pH 8: the pH dependence of EDTA as a preservative of high molecular weight DNA in biological samples Dear Dr. Distel: 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. Marco Bonizzoni Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0280807.r004

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