Oxford nanopore sequencing enables rapid discovery of single-domain antibodies from phage display libraries.
Biotechniques
; 65(6): 351-356, 2018 12.
Article
en En
| MEDLINE
| ID: mdl-30477332
ABSTRACT
Antibody (Ab) repertoire sequencing using high-throughput massively parallel technologies has contributed substantially to the understanding of Ab responses following infection, vaccination and autoimmunity. Because individual B-cell receptors are recombined and diversified somatically, genomic comparisons are limited, and distinguishing rare variants from sequencing errors is a major challenge. Oxford Nanopore Technologies' MinION is a highly portable and cost-effective third-generation sequencing instrument, but has not been used for Ab repertoire sequencing due to its high error rate (approximately 1/10 bases). Here, we applied nanopore sequencing to single-domain Ab (sdAb) repertoires and phage-displayed sdAb libraries. We show that despite low overall data fidelity, sdAb sequences could be reconstructed above a frequency threshold (â¼100 copies); however, distinguishing clonal sdAb variants was not always possible. The data quality was sufficient to enable rapid identification of antigen-specific sdAb sequences enriched during panning of phage display libraries, obviating the need for screening single clones.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Secuenciación de Nucleótidos de Alto Rendimiento
/
Anticuerpos de Dominio Único
/
Técnicas de Visualización de Superficie Celular
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Biotechniques
Año:
2018
Tipo del documento:
Article