Fig. 5

Bioinformatic analysis and selection of unique, library-specific clones for characterization using the IgX and SAbPred platforms. (a) The bioinformatics workflow for identifying unique clones from each library for IgG production and downstream characterization. In this scheme, each immune library was digested to generate DNA fragments containing the target scFv and submitted for long-read SMRT sequencing. Long-read outputs were then converted to a FASTQ format and, alongside FASTA-formatted Sanger data of previously screened hits, processed via MiXCR to generate paired reads. Using IgX Cluster and Branch, both Sanger and NGS data sets were clustered by sequence similarity and visualized as phylogenetic trees. Sequences from the NGS candidate pool were chosen based on similarity to well-characterized Sanger clones within the same cluster with validated function or binding. Chosen antibody sequences were then processed via IgX Track and promiscuous clones present in multiple libraries were removed to generate a final set of library-specific clones chosen for in silico mAb developability and canonical form prediction using TAP and SCALOP respectively. (b, c) Bar graphs showing (b) the number of clusters and (c) the average size of the clusters generated for each library-specific NGS data set using IgX Cluster. (d–g) V Gene and CDR3 length for chosen sequences from (d) the Target A paired, (e) the Target A combinatorial, (f) the Target B paired and (g) the Target B combinatorial libraries visualized as bubble plots. (h–k) Heat maps showing the sequence-based canonical form predictions for five CDRs—CDRL1, L2, L3 and CDRH1 and H2—in the bioinformatically chosen paired (h, j) and combinatorial (i, k) repertoires targeting Antigen A (h, i) and Antigen B (j, k). Scale bars indicate the percentage of chosen antibodies with each combination of predicted canonical forms.