Seq2scFv: a toolkit for the comprehensive analysis of display libraries from long-read sequencing platforms

Area of Science:

• Biotechnology and Pharmaceutical Sciences
• Computational Biology and Bioinformatics
• Immunology and Molecular Biology

Background:

• Antibodies are leading biotherapeutics, but traditional discovery methods are time-consuming and resource-intensive.
• High-throughput sequencing and computational methods advance antibody discovery by exploring a broader antibody space.
• Full-length sequencing of single-chain variable fragments (scFvs) from in vitro display libraries is a key advancement, yet tools for annotating paired heavy and light chain variable domains (VH and VL) are lacking.

Purpose of the Study:

• To introduce Seq2scFv, a novel open-source toolkit for analyzing in vitro display libraries using long-read sequencing data.
• To address the methodological gap in annotating paired VH and VL domains from full-length scFv sequences.
• To facilitate the efficient characterization of antibody display libraries and expedite the identification of high-affinity antibody candidates.

Main Methods:

• Development of Seq2scFv, an open-source toolkit for processing long-read sequencing data from in vitro display libraries.
• Utilizing Seq2scFv to identify and characterize V(D)J recombination in both VH and VL regions.
• Implementing functionalities for linker inference, sequence translation, numbering, unique identifier encoding, and quantification of identical sequences.

Main Results:

• Seq2scFv successfully annotates paired VH and VL domains from full-length scFv sequences.
• The toolkit provides translated sequences, numbered chains, and linker characterization.
• Seq2scFv enables sequence encoding with unique identifiers and quantification of sequence enrichment across selection rounds.

Conclusions:

• Seq2scFv addresses a critical methodological gap in antibody discovery by enabling comprehensive analysis of scFvs.
• The toolkit's versatile and standalone functionality simplifies the characterization of display libraries.
• Implementation of Seq2scFv is expected to accelerate the identification of high-affinity antibody candidates for biotherapeutic development.