Pipeline for Next Generation Sequencing data of phage displayed libraries to support affinity ligand discovery
Abstract: Affinity ligands are important molecules used in affinity chromatography for purification of significant substances from complex mixtures. To find affinity ligands specific to important target molecules could be a challenging process. Cytiva uses the powerful phage display technique to find new promising affinity ligands. The phage display technique is a method run in several enrichment cycles. When developing new affinity ligands, a protein scaffold library with a diversity of up to 1010-1011 different protein scaffold variants is run through the enrichment cycles. The result from the phage display rounds is screened for target molecule binding followed by sequencing, usually with one of the conventional screening methods ELISA or Biacore followed by Sanger sequencing. However, the throughput of these analyses are unfortunately very low, often with only a few hundred screened clones. Therefore, Next Generation Sequencing or NGS, has become an increasingly popular screening method for phage display libraries which generates millions of sequences from each phage display round. This creates a need for a robust data analysis pipeline to be able to interpret the large amounts of data. In this project, a pipeline for analysis of NGS data of phage displayed libraries has been developed at Cytiva. Cytiva uses NGS as one of their screening methods of phage displayed protein libraries because of the high throughput compared to the conventional screening methods. The purpose is to find new affinity ligands for purification of essential substances used in drugs. The pipeline has been created using the object-oriented programming language R and consists of several analyses covering the most important steps to be able to find promising results from the NGS data. With the developed pipeline the user can analyze the data on both DNA and protein sequence level and per position residue breakdown, as well as filter the data based on specific amino acids and positions. This gives a robust and thorough analysis which can lead to promising results that can be used in the development of novel affinity ligands for future purification products.
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