Oxford nanopore sequencing enables rapid discovery of single-domain antibodies from phage display libraries

• Published in: BioTechniques Vol. 65; no. 6; pp. 351 - 356
• Main Authors: Lowden, Michael J, Henry, Kevin A
• Format: Journal Article
• Language: English
• Published: England Future Science Ltd 01-12-2018; Taylor & Francis Group
• Subjects: Amino Acid Sequence; Animals; antibody; Base Sequence; Camelids, New World; Cell Surface Display Techniques - economics; Cell Surface Display Techniques - methods; H; High-Throughput Nucleotide Sequencing - economics; High-Throughput Nucleotide Sequencing - methods; Humans; nanopore sequencing; Nanopores; next-generation DNA sequencing; Peptide Library; phage display; Sequence Alignment; Single-Domain Antibodies - chemistry; Single-Domain Antibodies - genetics; single-domain antibody; Time Factors; V; VHH; nanopore sequencing; next-generation DNA sequencing; antibody; phage display; single-domain antibody; VHH
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/btn-2018-0123

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 (∼100 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.