Next Generation Sequencing
Specifica uses next generation sequencing (NGS) to assess the diversity of its libraries and analyze selection outputs.
The diversity of antibody libraries has traditionally been determined by counting the number of transformants, on the assumption that each bacterial clone contains a different antibody. With the advent of NGS it became possible to test that assumption. Upon sequencing a library developed by the CSO in 2000, it was found that instead of the expected HCDR3 diversity of ~100 million, the HCDR3 diversity was approximately thirty fold less (3 million). While the combined VH and VL diversity is significantly higher, due to the use of site specific recombination, the reduced diversity indicates the enormous scope for improvement in library construction, diversity, and functionality. Specifica uses NGS during library production to ensure diversity is maintained, and provides a minimal measure, as well as an estimate, of library diversity, using sophisticated in-house developed bioinformatics. Customers receive data files corresponding to antibody sequences obtained by NGS, which is useful in the analysis of selection outputs.
By applying NGS to selection outputs, Specifica is able to identify up to 5,000 different antibodies per target and bin these into different clonotypes, providing the broadest epitope coverage. Specifica has developed different methods to go from "sequence to clone".in order to provide clones from identified sequences.