Phage display antibody repertoire7/25/2023 By contrast, NGS approaches allow for far-greater insights into library diversity by providing up to 10 7 sequences (approximately 10,000-fold more sequences than Sanger sequencing). Although this approach is sufficient to identify dominant clones after selection, or to broadly validate design objectives, the data obtained represent only a limited snapshot of actual library diversity. Traditionally, antibody display libraries are analyzed by isolation of 10 2–10 3 clones in combination with Sanger sequencing ( 5). Strategies for NGS of Antibody Repertoires Here, we review recent advances in NGS technology and key applications to phage display and other in vitro selection technologies. While most NGS platforms were originally designed for short reads, technology is evolving rapidly, extending both read length and depth. This includes the sequencing of the paired human heavy and light chain repertoire from isolated naïve ( 8, 9) and antigen-specific B-cells ( 10, 11), as well as T-cell receptor ( 12) and antibody display repertoires ( 13). ![]() Although initially developed for genomics applications, such as whole-genome sequencing, transcriptome sequencing, and epigenetics, next-generation sequencing (NGS) technology is now increasingly being applied other fields, including to basic and applied immunology. Recent advances in DNA sequencing technologies and computing power over the last decade has led to a dramatic reduction in the cost of sequencing and has simplified data analyses ( 7). Consequently, clones with superior affinity may be present at low frequency and may not be readily detectable using traditional screening methods such as ELISA ( 6). Such clonal dominance can reflect genuine selection for high antigen affinity but might also reflect other properties such as superior expression or display. In many cases, later rounds of selections tend to be dominated by a handful of clones, which are then further characterized for affinity. Multiple rounds of selection are generally required to identify antigen-specific binders, either by binding to a solid support or through cellular sorting ( 5). The power of in vitro selection technologies relies on a direct physical link between phenotype (displayed antibody construct) and genotype (antibody variable domain genes), allowing for the identification of binders through sequencing of their encoding genes. These technologies bypass the use of animals and allow for the enrichment of binders within days to weeks. The development of antibody display technology such as phage ( 1), ribosome ( 2), yeast ( 3), and mammalian display ( 4) has enabled the rapid selection of binders from diverse libraries.
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