By-passing in vitro screening—next generation sequencing technologies applied to antibody display and in silico candidate selection

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 × 107 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.

Deep sequencing analysis of phage libraries using Illumina platform

“…The parsed files were then processed by quaseq.m script that assessed the quality of the R36 region containing the (NNK) 12 sequences. We selected only high-quality output in which all nucleotides had Phred Quality Score above 5 (this value could be changed in quaseq.m script on demand).…”

“…Illumina/Solexa deep-sequencing technology analyzes a library of blunt-ended double stranded DNA (dsDNA) fragments and generates up to 10 9 base pair (bp) reads in a single run. For example, Fisher and co-workers recently demonstrated the use of Illumina sequencing to characterize phage-displayed libraries of single chain antibodies (scFv) [12]. Fields and co-worker used Illumina sequencing to characterize selection from libraries of WW-protein displayed on T7 phage [13].…”

Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets

“…Those domains within the ScFv that have the most diversity, and form the centre of the antigen combining site, the CDR3 domains, have been the sequencing target of choice for in silico identification of enriched sequences. Methods for the analysis of the NGS datasets are either based on the comparison of clone frequency within the enriched sub libraries relative to the whole library (Ravn et al 2010) or are based on the comparison of positive and negative panning reactions (panning the same library against a target and an unrelated molecule) and the generation of Z-scores representing individual ScFv enrichment (Zhang et al 2012). Recently, Rebollo et al also described using the frequency of motif isolation within NGS datasets to identify enriched bicyclic peptide binders to different protein targets (Rebollo et al 2014).…”

Mining the antibodyome for HIV-1–neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains

“…Generally, only a few monoclonal antibodies from each donor have been identified, although it is possible to identify substantially more (6,12). Indeed, next-generation sequencing technologies (13)(14)(15)(16)(17)(18) seem to offer an efficient means for identifying thousands of somatic variants (19). The massively parallel sequencing that is used by such technologies, however, leads to the loss of information on individual pairings of heavy and light chain and as such, has made it a challenge to discern native antibodies (with naturally paired heavy and light chains) in next-generation sequencing data.…”