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Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing
Published on: October 3, 2018
Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective
Emmanuel Dias-Neto1, Diana N Nunes, Ricardo J Giordano
1David H. Koch Center, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.
Plos One
|December 19, 2009
Summary
We developed a faster, more accurate, and cost-effective method for phage display library screening using quantitative PCR and next-generation sequencing. This advanced technique improves ligand identification and analysis for various applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Genomics
Background:
- Combinatorial phage display is a key technique for identifying molecular recognition events, such as protein-ligand and protein-protein interactions.
- Traditional phage display library selection faces challenges with rate-limiting steps like counting transducing units and sequencing displayed ligands.
- Emerging genomic technologies offer potential solutions to overcome these limitations in phage display screening.
Purpose of the Study:
- To adapt and integrate emerging genomic technologies to streamline and enhance combinatorial phage display library selection.
- To develop a high-throughput quantitative and analytical toolset for more efficient phage display screening.
- To overcome the labor-intensive and time-consuming nature of traditional phage display methods.
Main Methods:
- Implemented real-time quantitative PCR (qPCR) for rapid and accurate quantification of phage particles.
- Utilized next-generation sequencing (NGS) for large-scale analysis of encoded ligands in phage display libraries.
- Integrated qPCR and NGS into a bacteria-free phage display screening workflow.
Main Results:
- The integrated approach significantly enhances efficiency, enabling bacteria-free phage display library screening.
- Achieved a >1,000-fold increase in the rate of phage display selection and ligand analysis.
- Reduced costs by approximately 250-fold for generating 10^6 ligand sequences, with increased sensitivity, accuracy, and scalability.
- Pyrosequencing results correlated well with conventional Sanger sequencing, showing no biases in sequence data.
Conclusions:
- The developed method, qPhage plus pyrosequencing, provides a faster, less expensive, and more accurate analysis of phage enrichment compared to traditional methods.
- This approach allows for significantly larger sampling sizes, leading to more robust and reliable results.
- qPhage plus pyrosequencing is proposed as a superior method of choice for a wide range of in vitro, in-cell, and in vivo phage display applications.
