Cloning antibodies from single cells in pooled sequence libraries by selective PCR

Felix Horns; Stephen R. Quake

doi:10.1371/journal.pone.0236477

Peer Review History

Original SubmissionMay 7, 2020
11 Jun 2020 Decision Letter - Kevin A. Henry, Editor PONE-D-20-13444 Cloning antibodies from single cells in pooled sequence libraries by selective PCR PLOS ONE Dear Dr. Horns, 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 Jul 26 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. 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To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Additional Editor Comments (if provided): [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: N/A Reviewer #3: 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 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: The manuscript by Horns and Quake presents an elegant method to retrieve monoclonal antibody sequences (VH and VL) from cDNA high-throughput sequencing data for subsequent production of recombinant antibodies. The method relies on the use of single-cell barcodes when performing single-cell sequencing to specifically amplify the antibody sequences of interest, by nested PCR. The authors first describe the computational methodology then moved onto a functional example from a 10x Genomics 5' V(D)J sequencing experiment. The manuscript is clearly written and will be helpful for a number of scientists performing single-cell antibody NGS using the 10X Genomics 5' V(D)J kit and who would like to further characterise some antibodies sequenced. Minor comments: (1) Did the paired heavy-light chain sequencing cover the whole VH and VL sequences or just some CDRs? If only some CDRs are being sequenced, would this mean that some framework (FR) regions be missed? In particular the FR1, where the primers for PCR2 will bind. (2) Why can SPAR primers be designed for only 88% of VH and 90% of VL? Can the barcodes misprime to other cDNA sequences? Or is it due to poor sequencing of the framework 1 or the VH or VL? (3) Why was the retrieval and cloning of the VH and VL so inefficient in the end? Figure 3E shows that between 1 and 3 out of 8 clones had 100% DNA sequence identity Reviewer #2: Summary: The authors describe an approach to recover specific antibody amplicons from a pool of bulk cDNA for cloning and expression. Overall the manuscript is clearly written and easy to follow. The introduction, however, emphasizes that functional evaluation of antibodies is a bottleneck, which is true, but the proposed approach only addresses one piece of the functional screen bottleneck, which is cloning. It's a bit overstated to imply that the proposed approach addresses the functional screen bottleneck. Additionally, while I appreciate the arguments being made, I find tables 1 and 2 as not adding major contributions to the paper. Table 1 for example doesn't include personnel time. The cost of ordering the heavy and light chain genes for synthesis is ~$100, so the cost argument isn't super strong given how easy it is to order genes these days. Second, Table 2 is a bit subjective - would 1 hr of hands on time for primer design be consistent for everyone? In many ways, it is much easier to just order the gene for synthesis. I think there's still value to the approach, however, for anyone who really wants to do an immediate PCR to recover HC+LC genes instead of waiting for gene synthesis, but I don't think this needs to be overhyped. I still think worth publishing in plos one. Reviewer #3: The authors report a very clever technique for rapidly extracting individual mAbs of interest from bulk 10x Genomics sequencing libraries. The manuscript is well written, the analyses and experiments are technically sound, and the data is clearly presented. I recommend acceptance and publication without any modification. ****** 6. 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Revision 1
25 Jun 2020 Author Response Response to editor comments 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We have revised the manuscript to conform to the style requirements. 2. 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.org if you have any questions. All original gel images are now included as S1_raw_images. These images were used to generate Figure 3B and Figure 3C. We are providing this image data as Supplementary Information. We confirm that the gels reported in the main figures were prepared according to the guidelines. Specifically, no manipulation of the images was performed except for cropping empty lanes. 3. We note that you have a patent relating to material pertinent to this article. Please provide an amended statement of Competing Interests to declare this patent (with details including name and number), along with any other relevant declarations relating to employment, consultancy, patents, products in development or modified products etc. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. This information should be included in your cover letter; we will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests We are preparing to submit a patent application related to this manuscript. We will provide an amended statement of Competing Interests as soon as the application is submitted and the patent name and number are available. This does not alter our adherence to PLOS ONE policies on sharing data and materials, and there are no restrictions on sharing of data or materials. We confirm that all authors do not have potential competing interests. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. We are happy to add the ORCID iDs for all authors. However, when attempting to link the ORCID iD in Editorial Manager, we received a notice “The ORCID site reported the following error and the authorization process cannot be completed:” (The rest of the error message was blank). The ORCID iD of corresponding author Stephen Quake is https://orcid.org/0000-0002-1613-0809, and the ORCID iD of author Felix Horns is https://orcid.org/0000-0001-5872-5061. Response to reviewers Referee 1 (1) Did the paired heavy-light chain sequencing cover the whole VH and VL sequences or just some CDRs? If only some CDRs are being sequenced, would this mean that some framework (FR) regions be missed? In particular the FR1, where the primers for PCR2 will bind. The paired heavy- and light-chain sequencing covers the entire VH and VL sequences. Thus, no framework regions are missed. We have added a sentence explaining this (Line 100). (2) Why can SPAR primers be designed for only 88% of VH and 90% of VL? Can the barcodes misprime to other cDNA sequences? Or is it due to poor sequencing of the framework 1 or the VH or VL? For ~10% of heavy- and light-chain genes, SPAR PCR primers satisfying constraints on predicted melting temperature, G/C content, and secondary structure could not be found. While we have considered these genes irretrievable, fine-tuning of PCR conditions might allow them to be retrieved. Additionally, if we used a library preparation method that incorporated a larger CBC and UMI, then there would be a higher likelihood that suitable SPAR PCR1 primers could be designed. We have added a comment on these sources of irretrievability and strategies to mitigate them in the Discussion (Line 183). (3) Why was the retrieval and cloning of the VH and VL so inefficient in the end? Figure 3E shows that between 1 and 3 out of 8 clones had 100% DNA sequence identity Nearly all of the clones without perfect DNA sequence identity to the target had suffered one or a few single base changes. We attribute this to the imperfect fidelity of the polymerases used for the various PCR steps. This issue could be mitigated by reducing the number of cycles of PCR or using higher fidelity polymerases. We have added a comment about this to the Discussion (Line 189). Referee 2 The introduction, however, emphasizes that functional evaluation of antibodies is a bottleneck, which is true, but the proposed approach only addresses one piece of the functional screen bottleneck, which is cloning. It's a bit overstated to imply that the proposed approach addresses the functional screen bottleneck. We agree that the functional evaluation of antibodies faces further bottlenecks downstream of cloning and expression. We have amended the main text in several places to emphasize that our method addresses the cloning bottleneck (Line 70 in the introduction and Line 162 in the discussion). Additionally, while I appreciate the arguments being made, I find tables 1 and 2 as not adding major contributions to the paper. Table 1 for example doesn't include personnel time. The cost of ordering the heavy and light chain genes for synthesis is ~$100, so the cost argument isn't super strong given how easy it is to order genes these days. We have clarified that the cost of SPAR is cheaper than or similar in price to gene synthesis (Line 174). Second, Table 2 is a bit subjective - would 1 hr of hands on time for primer design be consistent for everyone? In many ways, it is much easier to just order the gene for synthesis. I think there's still value to the approach, however, for anyone who really wants to do an immediate PCR to recover HC+LC genes instead of waiting for gene synthesis, but I don't think this needs to be overhyped. We agree that the speed of SPAR is a valuable aspect. We have edited the text to emphasize that the turnaround time is faster than gene synthesis and to highlight the situations where this might be advantageous (Line 176). With respect to Table 2, while it is true that the times are subjective, we believe that it is helpful for practitioners of the method to understand the approximate timelines. This is often presented in molecular biology methods papers. We also believe that these timelines, while approximate, illustrate that the timescale is substantially faster than gene synthesis. Nevertheless, we have now indicated in the Table legend that these times are approximate. Attachments Attachment Submitted filename: Rebuttal_200613.docx https://doi.org/10.1371/journal.pone.0236477.r002
9 Jul 2020 Decision Letter - Kevin A. Henry, Editor Cloning antibodies from single cells in pooled sequence libraries by selective PCR PONE-D-20-13444R1 Dear Dr. Horns, 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, Kevin A. Henry Academic Editor PLOS ONE Additional Editor Comments (optional): I am pleased to say that the reviewers have found the revised manuscript suitable for publication in PLoS One. Congratulations on the very nice work. Reviewer 2 contacted me by email to confirm that their concerns had been addressed. 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: All the comments have been properly addressed. The manuscript should be accepted as is, without any further improvements. ****** 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 https://doi.org/10.1371/journal.pone.0236477.r003
Formally Accepted
17 Jul 2020 Acceptance Letter - Kevin A. Henry, Editor PONE-D-20-13444R1 Cloning antibodies from single cells in pooled sequence libraries by selective PCR Dear Dr. Horns: 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. Kevin A. Henry Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0236477.r004

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