Single-cell measurement quality in bits

Jayan Rammohan; Swarnavo Sarkar; David Ross

doi:10.1371/journal.pone.0269272

Peer Review History

Original SubmissionNovember 15, 2021
18 Jan 2022 Decision Letter - Panayiotis V. Benos, Editor PONE-D-21-36062Single-cell measurement quality in bitsPLOS ONE Dear Dr. Ross, 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 pay attention to the comments of all reviewers and in particular reviewer #3. You need to address all their comments in the revision. Please submit your revised manuscript by Mar 04 2022 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No ****** 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 Reviewer #3: 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 Reviewer #3: 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: General comments: The manuscript is well written and understandable and conclusions in general appear well supported by the data presented in the manuscript. Overall I have two general points that could be added to the manuscript: 1) Data analyzed in this manuscript was published beforehand by the same laboratory (PMID:34079048), as the authors state. However, it would be helpful to summarize how the current analysis and its conclusions relate to what has been published before. 2) It appears that if different environmental inputs/stimuli would be used, channel capacity estimates could change. Is the expected in practice? Would it have the potential to affect observations reported, for example that for RNA microscopy has typically higher capacity than flow cytometry? If such a caveat exists, it would be good to point this out in the manuscript. Specific comments There are instances where capacity reported in the text is different from what is in the corresponding table. For instance, on page 11 the text quotes the capacity for HCR labeled RNA as 0.08 bits, whereas table 1 shows 0.09 bits; in the text the capacity of FISH-labeled RNA and microscopy is quoted as 0.97 bits, whereas table 1 shows 1.06 +/- 0.07 bits, which puts the value in the text outside of three standard deviations from what is in the table. It would be helpful, throughout the manuscript, if capacities in the text would match information given in tables. Reviewer #2: The authors use mutual information measure to compare different experimental methods of single cell expression analysis. It is a well-controlled analysis with a small number of variables. it isn't exactly clear how useful it will be for large scale experiments, such as scRNA-seq, but they state that it should be generalizable. Perhaps they will do that in future work. I do not find any technical problems with the work or the presentation so I conclude that it meets the PLoS One criteria for publication. Reviewer #3: In this article, the authors proposed an information theory approach for assessing and compare single-cell measurement quality in bits. They then claim that it provides a universally comparable metric for information content. 1- Although the article tackle a very important issue, the manuscript is not detailed enough. - More information and detail explanations between single-cell measurement and information theory should be given. - The methodology section on the computation of the channel capacity is about 5 lines with references to supporting materials where more details are given. I think these details should be part of the manuscript itself to allow the readers to have a flow. 2- In Table 1 and Table 2, what is the ground true? 3- In the support materials, page 2 the authors said: "This vector has the same dimension as the number of input concentrations, we chose an uniform discrete distribution in our work, i.e., p_j^0=0.125 for each j. ε - Numerical error value for convergence, we chose ε=〖10〗^(-4)." - More explanations should be given. Why a normal distribution, why 0.125 and 10^-4, how do the results vary relative to these parameters. A sensitivity analysis of these parameters should be performed. 4 - There are some texts similarities between this manuscript and the manuscript [reference 10] recently published by the same authors. Some of these parts should be rewritten. ****** 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 Reviewer #3: 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.0269272.r001
Revision 1
2 Mar 2022 Author Response We thank the editors and reviewers for the thoughtful reviews of our manuscript. We have made changes to the manuscript in response to each of the reviewers’ comments as detailed below. In general, we have tried to clarify the descriptions of our methods, and we have moved the content that was in the SI (detailed descriptions of the algorithms used) to the Materials and Methods section of the manuscript. Reviewer #1: General comments: The manuscript is well written and understandable and conclusions in general appear well supported by the data presented in the manuscript. Overall I have two general points that could be added to the manuscript: 1) Data analyzed in this manuscript was published beforehand by the same laboratory (PMID:34079048), as the authors state. However, it would be helpful to summarize how the current analysis and its conclusions relate to what has been published before. Response: In the second sentence of the introduction, we added text to more clearly summarize the relationship between the previous publication and the current manuscript: “… we used a recently-reported collection of data from different single-cell measurements… [10]. In that study, a split-sample approach was used to measure cells harvested from the same replicate cultures… Here, we use the experimental results from [10] to show how channel capacity can be used as a metric…” 2) It appears that if different environmental inputs/stimuli would be used, channel capacity estimates could change. Is the expected in practice? Would it have the potential to affect observations reported, for example that for RNA microscopy has typically higher capacity than flow cytometry? If such a caveat exists, it would be good to point this out in the manuscript. Response: Yes, different inputs/stimuli would result in different channel capacity estimates. We have added a new paragraph (page 5 of the revised manuscript) to make this point clear: “As a final consideration, it is important to note that the channel capacity can also depend on the choice of values used for the input stimuli. If the channel capacity is the logarithm of the number of distinguishable input levels, then it clearly cannot be greater than the logarithm of the number input levels measured. For example, if an experiment only uses two input levels (e.g., test and control, or high and low), then the channel capacity determined by our approach will always be less than or equal to one (log2(2) = 1). Furthermore, to obtain the best estimates of the biological channel capacity and the best comparison of different methods, the input levels should be chosen to span the full range of biological response. For example, the datasets used here include IPTG concentrations across the full induction curve, with input levels that result in low biological output (i.e., gene expression), high biological output, and intermediate biological output. In general, a different choice of input levels measured in an experiment could lead to different estimates of the channel capacity with our approach. So, for assessment of measurement quality, we recommend comparing only methods implemented with the same set of input levels (as with the split-sample dataset used here).” Specific comments There are instances where capacity reported in the text is different from what is in the corresponding table. For instance, on page 11 the text quotes the capacity for HCR labeled RNA as 0.08 bits, whereas table 1 shows 0.09 bits; in the text the capacity of FISH-labeled RNA and microscopy is quoted as 0.97 bits, whereas table 1 shows 1.06 +/- 0.07 bits, which puts the value in the text outside of three standard deviations from what is in the table. It would be helpful, throughout the manuscript, if capacities in the text would match information given in tables. Response: We thank the reviewer for pointing out these discrepancies. We have carefully compared the text and the tables to ensure the values match in the revised manuscript. We also re-checked the standard deviations used in the tables and found that we had mistakenly used the population standard deviation. So, we re-calculated the standard deviations reported in the table (using the sample standard deviation). Reviewer #2: The authors use mutual information measure to compare different experimental methods of single cell expression analysis. It is a well-controlled analysis with a small number of variables. it isn't exactly clear how useful it will be for large scale experiments, such as scRNA-seq, but they state that it should be generalizable. Perhaps they will do that in future work. I do not find any technical problems with the work or the presentation so I conclude that it meets the PLoS One criteria for publication. Response: We have added an additional sentence to the Discussion to briefly address the generalizability to measurements such as scRNA-seq (page 13 of the revised manuscript): “For more complex, multi-variate single-cell measurements (e.g., multi-transcript RNA-seq, time-series microscopy), application of our approach to compare different measurement protocols and/or data analysis methods would probably require an alternative to the Blahut-Arimoto algorithm for estimating the channel capacity [23]. Our general approach should still be valid for those types of data, however: the highest quality method will be the one that results in the highest channel capacity.” Reviewer #3: In this article, the authors proposed an information theory approach for assessing and compare single-cell measurement quality in bits. They then claim that it provides a universally comparable metric for information content. 1- Although the article tackle a very important issue, the manuscript is not detailed enough. - More information and detail explanations between single-cell measurement and information theory should be given. - The methodology section on the computation of the channel capacity is about 5 lines with references to supporting materials where more details are given. I think these details should be part of the manuscript itself to allow the readers to have a flow. Response: We agree with the reviewer and have moved the description of the Blahut-Arimoto algorithm and our specific implementation to the Materials and Methods section of the manuscript. We have also edited that text and the brief description of the approach in the Results section to more clearly describe the methods used. 2- In Table 1 and Table 2, what is the ground true? Response: The closest thing to “ground truth” in this case is the true biological channel capacity, which will be greater than the measured channel capacities. So, the best estimate of the biological channel capacity is the highest measured channel capacity (corresponding to the highest quality measurement method). We have edited the text (page 7 of the revised manuscript) to clarify this point: “Since no measurement is perfect, the measurement channel will degrade the information that it transmits. So, the measured channel capacity, i.e. the channel capacity between the input and the estimated gene expression, will always be less than the biological channel capacity, i.e., the channel capacity between the input and the actual gene expression. Higher quality measurements, however, will degrade the information less. So, we can assess relative measurement quality by comparing the measured channel capacities for different measurement methods: Higher quality measurements will result in a higher measured channel capacity. (i.e., closer to the true biological channel capacity).” 3- In the support materials, page 2 the authors said: "This vector has the same dimension as the number of input concentrations, we chose an uniform discrete distribution in our work, i.e., p_j^0=0.125 for each j. ε - Numerical error value for convergence, we chose ε=〖10〗^(-4)." - More explanations should be given. Why a normal distribution, why 0.125 and 10^-4, how do the results vary relative to these parameters. A sensitivity analysis of these parameters should be performed. Response: We have moved the relevant sections from the SI to the Methods section and revised them to improve the clarity. In particular, in the revised manuscript, we point out that the Blahut-Arimoto algorithm converges monotonically toward exact channel capacity value (as shown in the 1972 papers by Blahut and Arimoto), so the result does not depend on the choice of p_j^0. Furthermore, we explain that the parameter ε determines how close the iterative algorithm must get to the exact answer before the iterations are stopped. So, we chose ε (0.0001 bits) to be much small than the typical uncertainty in the channel capacity estimates (>= 0.03 bits, from measurement replicates, see manuscript Tables 1-3). 4 - There are some texts similarities between this manuscript and the manuscript [reference 10] recently published by the same authors. Some of these parts should be rewritten. Response: This point is difficult to address without more specific information from the reviewer (i.e., which parts of the manuscript have text too similar to the previous publication). However, we carefully compared the text from the two manuscripts, paying particular attention to paragraphs we thought might have overlap. Although the two manuscripts have some overlap in language (they are related manuscripts), we only found one instance of a string longer than four words that was identically repeated in both manuscripts; “… in parallel on cells harvested from the same original culture.” In the revised manuscript, we have modified the text to avoid using the same sentence fragment as in the previous publication. Attachments Attachment Submitted filename: Measurement_Quality_PLOS ONE.2.response to reviews.docx https://doi.org/10.1371/journal.pone.0269272.r002
18 May 2022 Decision Letter - Panayiotis V. Benos, Editor Single-cell measurement quality in bits PONE-D-21-36062R1 Dear Dr. Ross, 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, Panayiotis V. Benos, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0269272.r003
Formally Accepted
2 Aug 2022 Acceptance Letter - Panayiotis V. Benos, Editor PONE-D-21-36062R1 Single-cell measurement quality in bits Dear Dr. Ross: 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 Panayiotis V. Benos Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0269272.r004

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