Full length articleNext generation sequencing of all variable loops of synthetic single framework scFv-Application in anti-HDL antibody selections

•Method for sequencing all CDRs of scFv from a synthetic single framework library with Illumina MiSeq.•Simple software for analyzing the CDR sequences and read numbers of antibody clones.•Any antibody clone in NGS read-out can easily be retrieved by reproducing it from synthetic DNA.

Next generation sequencing (NGS) can be applied to monitoring antibody phage display library selection processes to follow the enrichment of each individual antibody clone. Utilising the recent development of the Illumina sequencing platform enabling sequencing up to 2 × 300 bp, we have developed a method to deep sequence all complementarity determining regions (CDRs) in the clones obtained from a synthetic single framework antibody library. This was complemented by an in-house bioinformatics pipeline for efficient analysis of the sequencing results. The method was utilised to study antibody selections against high density lipoprotein (HDL) particles. Sequencing of the output from each selection round enabled extraction of useful information on both the total copy numbers as well as the relative enrichment rates of the clones. Ten antibody clones showing different ranking in terms of frequency were reproduced from synthetic DNA constructs and their capacity to bind HDL was verified by an immunoassay. The method thus facilitates the isolation of clones of interest, and in particular can assist retrieval of less efficiently enriched, yet interesting clones, which are unlikely to be identified by conventional, random colony picking based, screening.