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European Journal of Immunology Supporting Information for DOI 10.1002/eji.201545526 Rajagopal Murugan, Katharina Imkeller, Christian E. Busse and Hedda Wardemann Direct high-throughput amplification and sequencing of immunoglobulin genes from single human B cells
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
Direct high-throughput amplification and sequencing of immunoglobulin genes from single human B cells Murugan, Rajagopal; Imkeller, Katharina; Busse, Christian; Wardemann, Hedda Correspondence: Prof. Wardemann, Hedda, Im Neuenheimer Feld 280, Heidelberg, 69120 __________________________________________________________________________________ Review Timeline:
Submission date:
28-Jan-2015
First Editorial decision:
02-Mar-2015
Revision/s received:
05-May-2015
Second Editorial decision:
01-Jun-2015
Final Revision received:
17-Jun-2015
Third editorial decision:
22-Jun-2015
Accepted:
29-Jun-2015
___________________________________________________________________________________
Handling Executive Committee member: Prof. Hans-Martin Jäck Please note that the correspondence below does not include the standard editorial instructions regarding preparation and submission of revised manuscripts, only the scientific revisions requested and addressed.
First Editorial Decision - 02-Mar-2015
Dear Prof. Wardemann,
Manuscript ID eji.201545526 entitled "High-throughput single cell amplification and sequencing of full-length human immunoglobulin heavy and light chain genes" which you submitted to the European Journal of Immunology has been reviewed. The comments of the referees are included at the bottom of this letter.
A revised version of your manuscript that takes into account the comments of the referees will be reconsidered for publication.
You should also pay close attention to the editorial comments included below. *In particular, please edit your figure legends to follow Journal standards as outlined in the editorial comments. Failure to do this will result in delays in the re-review process.*
Please note that submitting a revision of your manuscript does not guarantee eventual acceptance, and that your revision will be re-reviewed by the referees before a decision is rendered.
If the revision of the paper is expected to take more than three months, please inform the editorial office. Revisions taking longer than six months may be assessed by new referees to ensure the relevance and timeliness of the data.
Once again, thank you for submitting your manuscript to European Journal of Immunology and we look forward to receiving your revision.
Yours sincerely, Karen Chu
On behalf of Prof. Hans-Martin Jack
Dr. Karen Chu Editorial Office European Journal of Immunology e-mail:
[email protected] www.eji-journal.eu
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Reviewer: 1
Comments to the Author
- The authors fail to show any data or analysis of actual human antibody repertoires using their method. Without such data there is no validation of their method. It is suggested that they revise the manuscript to include such data.
- Also, they should compared their paired IGH-IGK/L sequencing data with recently published papers from Dekosky et. al, Nature Med, 2014. For example, are similar germline pairings observed with both methods?
- The authors should also have spike-in controls to validate that their paired sequencing returns accurate results following NGS and bioinformatic analysis. This can be done with an immortalized human B cell line
or transiently transfected HEK cells expressing IGH and IGK/L genes. This control would be important to know how many false positive pairings are generated using their method.
-
On lines 15-18: “..at the 5' end of exon 2 encoding the N-terminus of FWR1, a region that is
strongly conserved even in IGH-V genes with high loads of somatic hypermutations.―
The authors should have a citation for this as it is critical in the design of their method.
-
How is PCR efficiency being calculated in Fig. 1A-C, exactly how many single cells were tested
under each condition to determine this efficiency?
Reviewer: 2
Comments to the Author This paper describes high-throughput analysis of paired V genes from single human B cells by using the V gene amplification with two-dimensional bar-coded primers and next generation sequencing.
Lack of novelty Although this technology enables to analyze the human antibody repertories, authors just descried the V gene amplification efficiency by modifying primers. To demonstrate the power of this technology, authors need to show some scientific evidences, such as differences in V gene repertories among three healthy volunteers. The numbers of paired of V genes per analysis are too small (24x16 of each V genes per volunteers?) for repertoire analysis.
Insufficient explanation of figure legend It is hard for reader to understand the matrix of Fig. 1D without referring authors' previously published papers. More detailed explanations are required.
___________________________________________________
First Revision – authors’ response – 05-Mar-2015
Reviewer: 1
The authors fail to show any data or analysis of actual human antibody repertoires using their method. Without such data there is no validation of their method. It is suggested that they revise the manuscript to include such data.
We now provide a detailed repertoire analysis as part of the Supplementary Figure 2.
Also, they should compared their paired IGH-IGK/L sequencing data with recently published papers from Dekosky et. al, Nature Med, 2014. For example, are similar germline pairings observed with both methods?
As requested by the reviewer we performed a direct comparison of the germline IGH-IGK/L pairings. More than 95% of IGH-IGK/L pairs from our data were present in the data from Dekosky et. al, Nature Med, 2015, suggesting the robustness of the technique in sampling most frequently associated VH-VL pairs. We now state this in the text.
The authors should also have spike-in controls to validate that their paired sequencing returns accurate results following NGS and bioinformatic analysis. This can be done with an immortalized human B cell line or transiently transfected HEK cells expressing IGH and IGK/L genes. This control would be important to know how many false positive pairings are generated using their method.
Since our analysis is performed at single cell level false positive pairings can only be the result of misannotated primer barcodes. The barcodes and identification strategy has been validated previously (Busse et al. EJI). False positive pairings have not been observed when we compared Sanger Sequencing and NGS data obtained from the same cells.
On lines 15-18: “..at the 5' end of exon 2 encoding the N-terminus of FWR1, a region that is strongly conserved even in IGH-V genes with high loads of somatic hypermutations.” The authors should have a citation for this as it is critical in the design of their method.
We now clarify that this is our own observation obtained from the analysis of highly mutated IGH sequences from pathogen-reactive memory B cell antibodies. The data is now shown in Supplemental
Figure 1.
How is PCR efficiency being calculated in Fig. 1A-C, exactly how many single cells were tested under each condition to determine this efficiency?
To address this question we modified the Figure legend noting that each symbol in the plot represents the overall PCR efficiency of an experiment run with 192 or 384 sorted single cells.
Reviewer: 2
Although this technology enables to analyze the human antibody repertories, authors just descried the V gene amplification efficiency by modifying primers. To demonstrate the power of this technology, authors need to show some scientific evidences, such as differences in V gene repertories among three healthy volunteers. The numbers of paired of V genes per analysis are too small (24x16 of each V genes per volunteers?) for repertoire analysis.
As requested by both reviewers we now include a detailed repertoire analysis as part of Supplementary Figure 2. We agree with the reviewer that it may be interesting to determine the degree of V gene repertoire differences among healthy donors but we feel that addressing this question goes beyond the scope of this Technical Comment. Given the enormous repertoire diversity in-depth analyses of several thousands of cells from each donor will be required to draw definite conclusions on the natural variability between individual donors. Publication of our manuscript will allow a broader scientific community to apply the strategy for in-depth assessments of human antibody repertoires in health and disease.
Insufficient explanation of figure legend It is hard for reader to understand the matrix of Fig. 1D without referring authors' previously published papers. More detailed explanations are required.
We now provide a more detailed description of the matrix PCR in the main text.
Second Editorial Decision - 01-Jun-2015
Dear Prof. Wardemann,
You recently submitted your revised Manuscript ID eji.201545526.R1 entitled "High-throughput single cell amplification and sequencing of full-length human immunoglobulin heavy and light chain genes" to European Journal of Immunology. Your manuscript has been re-reviewed.
Unfortunately, based on the overall assessment of both reviewers, the manuscript will not improve existing methods already published from the same and others group. Therefore, the manuscript will have a low impact and therefore needs to be rejected (please also see the enclosed comments).
We regret to have to inform you about this decision. However, in view of the great pressure for space at present, only those papers that receive very favorable reviews are eligible for the European Journal of Immunology.
Thank you for considering European Journal of Immunology for the publication of your research. We hope that the outcome of this specific submission will not discourage you from submitting future manuscripts.
Please note that all files associated with this submission (i.e. the manuscripts files that you uploaded to Manuscript Central) but not the manuscript record itself will be deleted in 60 days. We will therefore not be able to enter into any specific queries regarding the manuscript after this date.
Yours sincerely, Katharina Schmidt
On behalf of Prof. Hans-Martin Jack
Dr. Katharina Schmidt
Editorial Office European Journal of Immunology,
[email protected]
Reviewer: 1
Comments to the Author The authors have now incorporated more detailed data in their manuscript by using human single cells human donors. While suitable as a technical comment, there is substantial lack of novelty given their own previous publication and work of other groups.
By only showing 1152 single cells from 3 donors, with only a 52% success rate of IGH, it is deceptive to call this a "high-throughput method" as indicated in the title. This means there are only on average 200 cells/pairings per donor, which could be accomplished at a similar price by conventional single-cell PCR followed by Sanger sequencing.
Reviewer: 2
Comments to the Author Although one of the purposes of this report is to cut costs for high-throughput analysis of Ig repertoire, the booster (two independent nested IGH PCRs) seems tedious and costly. Does this improvement meet author’s goal?
The reason of the low IGH amplification efficiency should be explained.
Minor points that should be addressed: Supplementary Figure 2; 7, 8. What does 7, 8 mean? Authors response – 17-Jun-2015 Dear Prof. Jäck, Dear Hans-Martin, After submitting a revised version of our manuscript “High-throughput single cell
amplification and sequencing of full-length human immunoglobulin heavy and light chain genes“ (ID eji.201545526), which to our understanding addressed all of the reviewers’ criticisms, we were informed that our manuscript “will not improve existing methods already published from the same and others group“ and thus does not qualify for publication in EJI. We are puzzled by this comment and would highly appreciate clarification of this criticism. As detailed in our reply to the reviewers’ comments state of the art single cell based strategies involve the use of 21 different primers in 3 independent IGH PCR reactions (Scheid et& al. Science 2011) or fail to obtain full-length Ig gene amplicons for direct antibody cloning (DeKosky et&al. Nat. Biotech. 2013). In contrast, we have established a strategy that requires only one primer to amplify even highly mutated human IGHV genes of all isotypes at about 10x reduced costs compared to conventional Sanger sequencing-based approaches and at the same time is fully compatible with direct antibody cloning. Over the last twelve months we have successfully used the described matrix PCR approach to analyze human Ig gene repertoires of tens of thousands of cells under various conditions. Importantly, after direct Ig gene cloning we have been able to look at the functional antibody properties. The biological questions that we aim to answer in the different projects are highly complex and we strongly feel that including such data in the present manuscript will go way beyond the scope of a technical comment. Thus, our intention was to make this powerful tool to perform fast and cost-effective in-depth analyses of human antibody repertoires available to the broader scientific community. Surprisingly, reviewer 1 states that our manuscript qualifies as a Technical Report (as which it was submitted), but at the same time comments on its’ lack of novelty and specifically criticizes the number of cells that we analyzed. As requested in his/her first review we included a detailed repertoire analysis in the revised version of the manuscript and compared the data to those published by DeKosky et&al.. The results are highly similar demonstrating that our approach is valid. Thus, how could the analysis of higher numbers of cells improve the quality of a Technical Comment? Please find below our point-by-point response to the precise criticisms of the revised version of the manuscript in which we address all additional concerns of both reviewers in full. We would highly appreciate if you could consider these points and look forward to your reply.
On behalf of all authors yours sincerely,
Hedda Wardemann
Third Editorial Decision - 22-Jun-2015
Dear Prof. Wardemann,
Thanks for sending this appeal including a revised version of your manuscript and a detailed explanation of its importance. Your manuscript was thoroughly re-evaluated by our Executive Committe and they finally recommended publication of your technical comment.
Therefore, it is a pleasure to provisionally accept your manuscript entitled "High-throughput single cell amplification and sequencing of full-length human immunoglobulin heavy and light chain genes" for publication in the European Journal of Immunology. For final acceptance, please follow the instructions below and return the requested items as soon as possible as we cannot process your manuscript further until all items listed below are dealt with.
Please note that EJI articles are now published online a few days after final acceptance (see Accepted Articles: http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1521-4141/accepted). The files used for the Accepted Articles are the final files and information supplied by you in Manuscript Central. You should therefore check that all the information (including author names) is correct as changes will NOT be permitted until the proofs stage.
We look forward to hearing from you and thank you for submitting your manuscript to the European Journal of Immunology.
Yours sincerely, Katharina Schmidt
on behalf of Prof. Hans-Martin Jack
Dr. Katharina Schmidt Editorial Office European Journal of Immunology e-mail:
[email protected] www.eji-journal.eu
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