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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Genes, Immunoglobulin*
  • Genetic Variation*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Immunoglobulins / genetics*
  • Mice
  • Sequence Analysis, DNA / methods*

Substances

  • Immunoglobulins