Reliable RNA-seq analysis from FFPE specimens as a means to accelerate cancer-related health disparities research

Mitchell J. Frederick; Dannelys Perez-Bello; Pedram Yadollahi; Patricia Castro; Alan Frederick; Andrew Frederick; Rashid A. Osman; Fonma Essien; Imelda Yebra; Ashley Hamlin; Thomas J. Ow; Heath D. Skinner; Vlad C. Sandulache

doi:10.1371/journal.pone.0321631

Peer Review History

Original SubmissionDecember 19, 2024
19 Dec 2024 Author Response https://doi.org/10.1371/journal.pone.0321631.r001
13 Jan 2025 Decision Letter - Kazunori Nagasaka, Editor PONE-D-24-53608Reliable RNA-seq analysis from FFPE specimens as a means to accelerate cancer-related health disparities researchPLOS ONE Dear Dr. Sandulache, 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 Feb 27 2025 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. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Dear Authors, Thank you for submitting your manuscript to PLOS One. Based on the comments from our reviewers, our decision is "Minor Revision." Please revise the manuscript accordingly, and we look forward to receiving your revised submission. Sincerely, Kazunori Nagasaka [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: No ****** 4. If the manuscript contains new data, have the authors made this data 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 article 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 highlight any specific errors that need correcting in the box below. 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: In this submitted protocol authors optimized guidelines for DNA and RNA purification from FFPE oropharynx carcinoma tissue, followed by RNA-seq and DNA SNP analysis. Introduction The rational background of the protocol establishment, the given requirements are properly described in the Introduction. Results The provided protocol with the details included in supplementary documents is a gap-filling requirement. The presented results are well-summarized and logically described. A correct and critical comparison to TCGA data is provided, discussed, and reasons for cross-dataset correlations failures were explained. Discussion The Discussion highlights the main points in this lab method process and provides suggestions. This submitted material is a great contribution to the research in the corresponding field, but also valuable for interdisciplinary use. Reviewer #2: Large collection of FFPE archival biopsies from hospitals and research institutes are valuable samples for generating transcriptome and exome data. The authors have provided a detailed protocol to isolate RNA and DNA from FFPE samples, generate RNA-seq and SNP array data, and perform bioinformatics to remove outlier samples and normalize expression levels between samples. Although they do not develop any new methods, the authors provide an end-to-end protocol from RNA/DNA isolation to bioinformatics analysis by combining existing methods with some modifications. The protocol may be useful to researchers without prior experience working with FFPE samples. Major deficiency of the described bioinformatics method is that it does not take into consideration the impact of the chemistry of formalin fixation on the sequences. It is well known that formalin fixation primarily causes modification of amino groups on the four bases (A, C, G, U) with differing probabilities and can impact the GC content of the sequenced reads. Also, the decreased gene coverage in RNA-seq (described as dropouts by the authors) is not completely random and is very different from the dropouts observed in single-cell RNA-seq. Thus, it is essential that QC checks specific to FFPE, especially formalin overfixation, is necessary. The method described by the authors may work when majority of the samples produce good quality data and a small proportion of outlier samples need to be identified, but not when a large proportion of samples have QC issues due to fixation protocol or FFPE storage issues. Please include additional QC steps that are specific to FFPE samples and steps to identify formalin overfixation. ****** 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: Yes: Jozsef Dudas 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.0321631.r002
Revision 1
17 Feb 2025 Author Response Response to reviewer comments: Reviewer #1: This submitted material is a great contribution to the research in the corresponding field, but also valuable for interdisciplinary use. Response: Thank you. Reviewer #2: Major deficiency of the described bioinformatics method is that it does not take into consideration the impact of the chemistry of formalin fixation on the sequences. It is well known that formalin fixation primarily causes modification of amino groups on the four bases (A, C, G, U) with differing probabilities and can impact the GC content of the sequenced reads. Also, the decreased gene coverage in RNA-seq (described as dropouts by the authors) is not completely random and is very different from the dropouts observed in single-cell RNA-seq. Thus, it is essential that QC checks specific to FFPE, especially formalin overfixation, is necessary. The method described by the authors may work when majority of the samples produce good quality data and a small proportion of outlier samples need to be identified, but not when a large proportion of samples have QC issues due to fixation protocol or FFPE storage issues. Please include additional QC steps that are specific to FFPE samples and steps to identify formalin overfixation. Response: We appreciate the astute observation and suggestions and agree completely. We now include a newly created tool called “GCplotQC” developed to analyze GC-bias in individual samples and quantitatively compare deviation from a reference standard like TCGA data or the cohort average. The tool graphically generates smoothed curves from plots of %GC content verses gene expression and calculates the area of deviation compared to a designated standard reference. The area of deviation for all samples is then outputted to a table for easy reference, while the curves for individual samples are stored as png images and aggregated as a PDF file. This can be used to gauge overall GC bias among the entire cohort or individually identify samples that have steeper GC bias presumably stemming from over fixation. We have now added two paragraphs to the Results section that describe the impact of GC-bias in our study, how that relates to gene dropout, and the differences we observed between the three library preparations used. These results are illustrated in three brand new supplementary figures now added (Supplementary Figures 10, 11, and 12). We also added an additional paragraph to the Discussion section that outlines the utility of the QC tool, and references bioinformatic techniques developed by others that could be applied to correct for GC-bias at various steps in the analysis. To more specifically answer the reviewer’s questions, we agree that the zero dropout we observed was not through the same mechanisms found for single cell RNAseq because the latter impacts samples randomly and our zero dropouts occurred uniformly across samples. This is now explained better in our modified Results section. We find that zero dropout in our cohorts was driven by both low expected gene expression and to some degree GC content. However, in our case the GC bias was more likely systemic and uniform across samples within a cohort that likely reflects a general limitation of formalin fixation. Indeed, this is why our sample processing protocol includes an elevated temperature incubation to reverse and mitigate the effects of fixation. We report that the GC-bias was much more pronounced for the TruSeq samples than for those prepared with the Stranded or MACE-Seq libraries. As for how much GC-bias is too much before an analytical correction should be applied, we leave that to the investigator to determine for themselves. Our tool will allow quantitation and visualization of the affect (e.g. QC), which can be used in decisions governing whether to utilize one of the many GC correction pipelines already available. The use of a reference standard like TCGA data should help detect whether some samples are over fixed, or the entire cohort is bad, and our methods employ a quantitative metric derived from the area between a sample’s curve and the curve generated from a chosen standard. We are very grateful for the reviewer’s challenging question and believe addressing the concern has significantly improved our manuscript and the utility of our published pipeline. https://doi.org/10.1371/journal.pone.0321631.r003
10 Mar 2025 Decision Letter - Kazunori Nagasaka, Editor Reliable RNA-seq analysis from FFPE specimens as a means to accelerate cancer-related health disparities research PONE-D-24-53608R1 Dear Dr. Sandulache, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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I am pleased to inform you that, after careful consideration, our expert reviewers have recommended your paper for acceptance in PLOS ONE. We believe your research makes a clear and valuable contribution to the clinical field, and we anticipate that your findings will be widely recognized and cited in future studies. Congratulations once again! We look forward to seeing your published work and hope you continue your excellent research. Sincerely, Kazunori Nagasaka Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data 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 #2: Yes ****** 5. Is the article 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 highlight any specific errors that need correcting in the box below. 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 #2: Authors have updated the manuscript to address the GC bias caused by formalin fixation. Although the reason for the GC bias is not adequately or accurately described, the authors describe it more as observations and should be adequate for the protocol reporting. Authors have provided code and ways to potentially detect or minimize GC bias. ****** 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 #2: No ******** https://doi.org/10.1371/journal.pone.0321631.r004
Formally Accepted
Acceptance Letter - Kazunori Nagasaka, Editor PONE-D-24-53608R1 PLOS ONE Dear Dr. Sandulache, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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 Kazunori Nagasaka Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0321631.r005

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