High-quality full-length immunoglobulin profiling with unique molecular barcoding

Abstract

High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.

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Figure 1: Base quality score heatmaps across the template length for three data pre-processing strategies.
Figure 2: Logic of the asymmetric paired-end sequencing strategy and data analysis with UMIs.
Figure 3: Replicate sample analysis.
Figure 4: Scheme of the cDNA library preparation and sequencing (Steps 6–22).
Figure 5: Required cDNA sequencing coverage.

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Acknowledgements

This work was supported by Russian Science Foundation project no. 14-14-00533. The work was carried out in part using equipment provided by the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Core Facility (CKP IBCH). M.S. is supported by an individual fellowship (mol-a-dk RFBR grant 16-34-60179). A.D., V.B., K.P. and S.P. are supported by the Ministry of Education, Youth and Sports of the Czech Republic (CEITEC 2020, LQ1601). K.P., V.B. and S.P. are supported by Ministry of Health, Czech Republic (AZV 15-30015A).

Author information

M.A.T., O.V.B., V.B., V.I.K., E.M.M., I.Z.M. and K.P. prepared the cDNA libraries and worked on the protocol. E.S.E., D.B.S. and O.K. worked on cell sample preparation. A.D. and M.D.L. worked on sequencing. M.A.T., O.V.B. and D.M.C. designed the experiments. A.D., M.S., D.A.B., M.I., S.P. and D.M.C. worked on data analysis and manuscript preparation.

Correspondence to D M Chudakov.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Title: Experimental datasets. Examples of full-length IGH profiling for the samples of human naive, plasma and memory B cells (XLSX 11235 kb)

Supplementary Table 2

Title: Control datasets. Full-length IGH profiling for the control set of CLL clones (XLSX 50 kb)

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Turchaninova, M., Davydov, A., Britanova, O. et al. High-quality full-length immunoglobulin profiling with unique molecular barcoding. Nat Protoc 11, 1599–1616 (2016). https://doi.org/10.1038/nprot.2016.093

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