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Nat Protoc. 2018 Jun;13(6):1232-1252. doi: 10.1038/nprot.2018.021. Epub 2018 May 3.

Unbiased quantification of immunoglobulin diversity at the DNA level with VDJ-seq.

Author information

1
Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK.
2
Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham Research Campus, Cambridge, UK.
3
Bioinformatics Group, The Babraham Institute, Babraham Research Campus, Cambridge, UK.

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.

PMID:
29725123
DOI:
10.1038/nprot.2018.021

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