1. By-passing in vitro screening—next generation sequencing technologies applied to antibody display and in silico candidate selection
- Author
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U. Ravn, Loïc Baerlocher, G. Magistrelli, M. Desmurs, N. Fischer, P. Malinge, Magne Osteras, M.H. Kosco-Vilbois, Laurent Farinelli, and F. Gueneau
- Subjects
Genetics ,Quality Control ,Sequence analysis ,In silico ,chemical and pharmacologic phenomena ,Enzyme-Linked Immunosorbent Assay ,Sequence Analysis, DNA ,Biology ,respiratory system ,Genome ,Complementarity Determining Regions ,Polymerase Chain Reaction ,DNA sequencing ,Cell Line ,Peptide Library ,Methods Online ,Humans ,Genomic library ,Peptide library ,Illumina dye sequencing ,Selection (genetic algorithm) ,Gene Library ,Single-Chain Antibodies - Abstract
In recent years, unprecedented DNA sequencing capacity provided by next generation sequencing (NGS) has revolutionized genomic research. Combining the Illumina sequencing platform and a scFv library designed to confine diversity to both CDR3, >1.9 × 10(7) sequences have been generated. This approach allowed for in depth analysis of the library's diversity, provided sequence information on virtually all scFv during selection for binding to two targets and a global view of these enrichment processes. Using the most frequent heavy chain CDR3 sequences, primers were designed to rescue scFv from the third selection round. Identification, based on sequence frequency, retrieved the most potent scFv and valuable candidates that were missed using classical in vitro screening. Thus, by combining NGS with display technologies, laborious and time consuming upfront screening can be by-passed or complemented and valuable insights into the selection process can be obtained to improve library design and understanding of antibody repertoires.
- Published
- 2010