Sequencing Antibody HCDR3 Regions

The plasmid DNA of the anti-CDK2 second sort output was used as a template for the PCR targeting the HCDR3 region of the scFvs. A set of forward primers mapping to the framework region upstream of the HCDR3 and carrying one of the Ion Torrent sequencing adaptors were used in combination with a barcoded reverse primer mapping to the common SV5 tag region of the yeast display vector and carrying the second adaptor required for sequencing. The primer sequences and method are described in detail by D’Angelo et al. (61 (link)). Once amplified with the proofreading Phusion polymerase (NEB), gel extracted, and quantified (Q-bit, HS-DNA kit, Invitrogen), the amplicon libraries were processed using the Ion Xpress Amplicon library protocol and then prepared for sequencing on the Ion 316 Chip (Life Technologies). The sequences analysis was performed using the AbMining Toolbox as described by D’Angelo et al. (61 (link)).