Unbiased quantification of immunoglobulin diversity at the DNA level with VDJ-seq.
Nat Protoc
; 13(6): 1232-1252, 2018 06.
Article
em En
| MEDLINE
| ID: mdl-29725123
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Variação Genética
/
Imunoglobulinas
/
Genes de Imunoglobulinas
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Análise de Sequência de DNA
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Nat Protoc
Ano de publicação:
2018
Tipo de documento:
Article