Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses

Dominika Borowska; Richard Kuo; Richard A. Bailey; Kellie A. Watson; Pete Kaiser; Lonneke Vervelde; Mark P. Stevens

doi:10.1371/journal.pone.0225658

Peer Review History

Original SubmissionAugust 12, 2019
11 Oct 2019 Decision Letter - Ruslan Kalendar, Editor PONE-D-19-22735 Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses PLOS ONE Dear Dr Borowska, 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. Authors should prepare reasoned comments for Reviewer #1 and make the necessary changes to the manuscript. We would appreciate receiving your revised manuscript by Nov 25 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Ruslan Kalendar, PhD Academic Editor PLOS ONE Journal Requirements 1. When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.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. Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Reviewer #1: This is a well written and justified presentation of a new method for analyzing chicken immune responses where other reagents are lacking. While this new method relies on analysis of RNA expression, this is an adequate first step when bulk protein analysis cannot be conducted due to limited reagents. Reviewer #2: The manuscript by Borowska et al., describes the generation of a high-throughput qPCR analysis approach for measuring 89 chicken immune genes. The genes were selected from a series of published RNA seq papers, and differentially expressed genes were identified. RNA samples were made from a series of isolated and challenged tissues, and cell types (bone marrow derived macrophages, dendritic cells, etc. ) PCR products were cloned and sequenced to validate the primer pairs for each gene. Primers spanned an intron where possible. The multiplex PCR was validated by examining the differential gene expression between a breeder farm and a sibling test farm (different biosecurity level). This is an extremely valuable tool for measuring chicken immune responses to pathogens. While the PCR products were validated individually, it is not clear to me that the same products would only be produced in the PCRs involving the primer mixes, where many more things could potentially interfere. I would encourage the authors to further validate this primer set prior to publication. I do not understand the rationale for the comparison of the two farms. While this study is interesting and potentially valuable to the industry partners, this would seem to have too many uncontrolled variables to actually be useful to test the 96.96 Fluidigm approach. I would have much preferred to examine the original RNAs used to identify the DE genes (agonist stimulated DC or M). Are these birds genetically related? Are they siblings? Is something known of their exposure to various pathogens. Any information would be helpful. Fig. 6 One of the three genes in the test by qPCR does shows the same pattern In Table 1. Gene name is probably RSAD1 ******** 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. Reviewer #1: Yes: Charles T Spencer Reviewer #2: No 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0225658.r001
Revision 1
7 Nov 2019 Author Response Dear Editor and Reviewers, Thank you for your email and the opportunity to revise our manuscript entitled ‘Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses’ (PONE-D-19-22735). We wish to thank the reviewers for their careful reading of this manuscript and for their constructive suggestions and comments. We have revised the manuscript accordingly and consider that the manuscript is improved as a consequence. Our response to the specific comments raised by each reviewer are detailed below, with the reviewer’s comments in italic type font and all references to text line numbers in parentheses are to the revised manuscript. As requested we have used track changes in the manuscript to indicate where the changes have been made and these can be found in the document ‘Revised Manuscript with Track Changes’. Journal requirements: 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. Where we indicated ‘data not shown’ in the original submission we have now removed the statement or provided additional information in the revised manuscript as follows: - Line 287 – 288 removed - Line 291 information added - Line 299 information removed - Line 411 supplementary figure added Reviewer #1: This is a well written and justified presentation of a new method for analyzing chicken immune responses where other reagents are lacking. While this new method relies on analysis of RNA expression, this is an adequate first step when bulk protein analysis cannot be conducted due to limited reagents. Thank you for a positive assessment. Your recognition of our work is much appreciated. Reviewer #2: The manuscript by Borowska et al., describes the generation of a high-throughput qPCR analysis approach for measuring 89 chicken immune genes. The genes were selected from a series of published RNA seq papers, and differentially expressed genes were identified. RNA samples were made from a series of isolated and challenged tissues, and cell types (bone marrow derived macrophages, dendritic cells, etc.) PCR products were cloned and sequenced to validate the primer pairs for each gene. Primers spanned an intron where possible. The multiplex PCR was validated by examining the differential gene expression between a breeder farm and a sibling test farm (different biosecurity level). This is an extremely valuable tool for measuring chicken immune responses to pathogens. While the PCR products were validated individually, it is not clear to me that the same products would only be produced in the PCRs involving the primer mixes, where many more things could potentially interfere. I would encourage the authors to further validate this primer set prior to publication. We have incorporated multiple steps to ensure that the primer pairs used in this study are most likely to be specific for one gene and not produce multiple products when applied in a mixture. Firstly, the primer pairs used in this study are gene-specific and have been carefully designed following the guidelines, as described in “Primer design for qPCR section”, lines 172 – 178. At least one of the primer (forward or reverse) was designed to span exon-intron boundary, in case of multi-exon genes, as described in the S3 Table. Secondly, the melting curve analysis was performed on each of the primer pairs in every reaction and only a single peak was detected regardless of the template sample used (cDNA or pre-amplified cDNA). If the primers were to align to non-specific sequences that would be detected in the melting plots as an additional peak or peaks relative to the target amplicon. Finally, the cDNA samples were pre-amplified with the primer mixture prior to the use on 96.96 Dynamic Array. If non-specific amplification occurs then this will not be repeated on the array because individual primer pairs are used in the separate chambers, amplifying only one gene. The DNA melting profile of each individual chamber on the array is indicative for its specificity and therefore we know that only specific products were amplified in the reactions. I do not understand the rationale for the comparison of the two farms. While this study is interesting and potentially valuable to the industry partners, this would seem to have too many uncontrolled variables to actually be useful to test the 96.96 Fluidigm approach. I would have much preferred to examine the original RNAs used to identify the DE genes (agonist stimulated DC or M). Are these birds genetically related? Are they siblings? Is something known of their exposure to various pathogens. Any information would be helpful. The gene list was designed to fit both purposes, to study primary cell cultures as well as a tool to screen performance of flocks raised under different environments. The decision was made to test the gene panel within the 96.96 Dynamic Array on samples collected from genetically related broilers that were housed in two different environments: a pedigree farm – representing a highly biosecure environment where breeding-programme selection candidates are recorded and selected, and the sibling-test farm - a non-bio-secure environment aimed to resemble broader commercial conditions and where full-siblings and half-siblings of selection candidates are placed. The vaccination schedule is described in lines 187-190. The farms differ greatly in microbial load but the exact composition of microbial community in the litter was not tested during this study. The bone marrow derived dendritic cells, heterophils and macrophages were derived from J line Brown Leghorn layers. This is an outbred population housed in National Avian Research Facility under conventional farm settings. The vaccination schedule of the J line chicken is described in lines 105-109. Additional pathogens may be present in the litter but were not tested during this study. Fig. 6 One of the three genes in the test by qPCR does shows the same pattern The profile of expression detected by qPCR is consistent with that observed using 96.96 Dynamic Array. We agree that only one gene tested in conventional qPCR has a significant difference in expression between farms, however the pattern is the same for all of the genes. In Table 1. Gene name is probably RSAD1 The gene symbol in the table 1 does not refer to the RSAD1 gene (radical S-adenosyl methionine domain containing 1). The gene symbol is correct - RASD1 ras related dexamethasone induced 1, NM_001044636, ENSGALG00000004860, as described in S3 Table. We are grateful for your editorial assistance and will be pleased to clarify any aspects. Yours sincerely Dr Dominika Borowska, The Roslin Institute & Royal (Dick) School of Veterinary Studies, University of Edinburgh. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0225658.r002
11 Nov 2019 Decision Letter - Ruslan Kalendar, Editor Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses PONE-D-19-22735R1 Dear Dr. Borowska, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Ruslan Kalendar, PhD Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0225658.r003
Formally Accepted
19 Nov 2019 Acceptance Letter - Ruslan Kalendar, Editor PONE-D-19-22735R1 Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses Dear Dr. Borowska: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ruslan Kalendar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0225658.r004

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