Abstract
For high-throughput sequencing and quantification of immunoglobulin repertoires, most methodologies use RNA. However, output varies enormously between recombined genes due to different promoter strengths and differential activation of lymphocyte subsets, precluding quantitation of recombinants on a per-cell basis. To date, DNA-based approaches have used V gene primer cocktails, with substantial inherent biases. Here, we describe VDJ sequencing (VDJ-seq), which accurately quantitates immunoglobulin diversity at the DNA level in an unbiased manner. This is accomplished with a single primer-extension step using biotinylated J gene primers. By addition of unique molecular identifiers (UMIs) before primer extension, we reliably remove duplicate sequences and correct for sequencing and PCR errors. Furthermore, VDJ-seq captures productive and nonproductive VDJ and DJ recombination events on a per-cell basis. Library preparation takes 3 d, with 2 d of sequencing and 1 d of data processing and analysis.
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Acknowledgements
We are grateful for invaluable assistance provided by K. Tabbada, Babraham Sequencing Facility; A. Davis, Babraham Flow Facility; and staff at the Babraham Biological Support Unit. This work was supported by the Biotechnology and Biological Sciences Research Council (BBS/E/B/000C0404), including a BBSRC Industrial CASE PhD studentship award to P.C. (1520397).
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The original version of the VDJ-seq assay was devised for mouse Igh by A.L.W. and D.J.B., and was adapted for human Igh and mouse Igδ by L.S.M. The improvements reported here were developed by P.C. and D.J.B. The original Babraham LinkON pipeline was developed by F.K. and S.A., and improvements and add-ons reported here were devised by P.C. Assay QC and supporting analyses were performed by P.C. P.C., D.J.B. and A.E.C. wrote the manuscript.
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A.L.W., D.J.B., L.S.M. and A.E.C. are named inventors on a patent, “Covering the VDJ-seq technique: Method of identifying VDJ recombination products” (UK Patent Application No. GB1203720.6, filed March 2, 2012; PCT Patent Applic. No. PCT/GB2013/05056, published September 6, 2013). National applications have been filed in Europe, the United States and Japan. US Publication number: 20150031042, publication date: January 29, 2015. The other authors declare no competing interests.
Integrated supplementary information
Supplementary Figure 1 VDJ-seq V gene usage across the mouse IgH locus compared to two other DNA-based methods.
Double width bars highlight V-genes that have been captured by VDJ-seq, but were not present using the other methods. (a) V-J primer cocktail method (Kaplinsky et al.1) compared to VDJ-seq. (b) HTGTS-Rep-seq compared to VDJ-seq. IGHV1-62-2 is missing in the HTGTS-Rep-seq data, but we believe this is because of IGHV1-62-2 and IGHV1-71 having identical sequence and filtering of multi-V gene calls would have removed these. All mice used for data generation were maintained in accordance with Babraham Institute Animal Welfare and Ethical Review Body and Home office rules under Project Licence 80/2529. Appropriate ARRIVE guidelines have been followed.
Supplementary Figure 2 Further comparison of VDJ-seq with the V-J primer cocktail method and HTGTS-Rep-seq.
(a) HTGTS-Rep-seq has a high correlation (r2=0.89-0.93) of V gene usage to our VDJ-seq method when comparing similar starting material. (b) Next, we chose to compare the number of clonotypes captured. We chose to compare clonotypes due to the high duplicate rate found in HTGTS-Rep-seq data, which we interpreted as PCR duplicates, as naïve spleen B-cells should not have high clonality. We found that VDJ-seq captured 2-5 times more clonotypes than HTGTS-Rep-seq. (c) Finally, we compared our method to using the classical cocktail of V-J primers from Kaplinsky et al.1. The correlation between the V gene usage from our method and using the V-J primer cocktail method is low (r2=0.58). All mice used for data generation were maintained in accordance with Babraham Institute Animal Welfare and Ethical Review Body and Home office rules under Project Licence 80/2529. Appropriate ARRIVE reporting guidelines have been followed.
Supplementary Figure 3 Impact of chew back on J gene mispriming correction.
The seqlogos illustrate the extent of chew back experienced by the J genes from NHEJ. Bases highlighted in bold capitals are the 5bp used for mispriming correction. It is possible to use these 5bp for mispriming correction as they are either after the primer sequence in the case of J2 and J3 or the end of the original primer sequence in the case of J1 and J4 remains after other J primers misprime. The chew back is most prominent at the site of cleavage (at the heptamer sequence (blue) of the recombination signal sequence (RSS)) and decreases in effect as a function of distance. This is illustrated by the gradual transition of J1 and J4. J2 and J3 have a much sharper transition due to the proximity of the 5bp to the cleavage site (5bp for J2 and J3 compared to 13bp for J1 and 11bp for J4). This means the 5bp used for mispriming correction will be conserved less often for J2 and J3 then they will be for J1 and J4. The J gene sequence underneath the seqlogo's illustrates the position of the J primers (red) within the J gene along with the 5bp used for mispriming correction. All mice used for data generation were maintained in accordance with Babraham Institute Animal Welfare and Ethical Review Body and Home office rules under Project Licence 80/2529. Appropriate ARRIVE reporting guidelines have been followed.
1. Kaplinsky, J. et al. Antibody repertoire deep sequencing reveals antigen-independent selection in maturing B cells. Proc. Natl. Acad. Sci. 111, E2622–E2629 (2014).
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Chovanec, P., Bolland, D., Matheson, L. et al. Unbiased quantification of immunoglobulin diversity at the DNA level with VDJ-seq. Nat Protoc 13, 1232–1252 (2018). https://doi.org/10.1038/nprot.2018.021
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DOI: https://doi.org/10.1038/nprot.2018.021
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