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Developing a Nanopore Sequencing Workflow for Protein Engineering Applications.

Daniel Zhi Jun Tan1, Vincent Fung1, Tao Sun1

  • 1Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117585, Singapore.

ACS Synthetic Biology
|July 5, 2023
PubMed
Summary
This summary is machine-generated.

Nanopore sequencing significantly reduces strand exchange errors in protein engineering mutant libraries compared to Illumina sequencing. An optimized nanopore workflow aids in identifying highly active alcohol dehydrogenase mutants.

Keywords:
Escherichia coligrowth-based selectionnanopore sequencingprotein engineeringsecondary alcohol dehydrogenasesite-saturated mutagenesis

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Protein Engineering

Background:

  • Next-generation sequencing (NGS) is crucial for identifying genetic mutations in protein engineering.
  • Commercially available NGS technologies like Illumina and nanopore sequencing have different error profiles.
  • Strand exchange is a known issue in sequencing mutant libraries, potentially confounding results.

Purpose of the Study:

  • To evaluate and compare the performance of Illumina and nanopore sequencing for analyzing protein engineering mutant libraries.
  • To develop and optimize a library preparation workflow for nanopore sequencing to minimize strand exchange.
  • To demonstrate the utility of the optimized nanopore workflow in identifying improved protein variants.

Main Methods:

  • Comparative analysis of Illumina NGS and nanopore sequencing on existing and in-house constructed mutant libraries.
  • Development of a novel library preparation workflow for nanopore sequencing.
  • Application of the optimized workflow for growth-based selection of alcohol dehydrogenase mutants.
  • Quantification of mutant enrichment fold change using nanopore sequencing data.

Main Results:

  • Illumina sequencing showed a substantial proportion of reads with strand exchange, mixing mutant information.
  • Nanopore sequencing demonstrated a significant reduction in strand exchange compared to Illumina.
  • The optimized nanopore library preparation workflow further decreased strand exchange incidence.
  • The workflow successfully quantified enrichment fold changes for 1728 mutants during selection.
  • A highly active alcohol dehydrogenase mutant (>500% improvement) was identified using fold change data.

Conclusions:

  • Nanopore sequencing offers a more accurate method for analyzing protein engineering mutant libraries due to reduced strand exchange.
  • The developed nanopore library preparation workflow enhances accuracy and efficiency in protein engineering.
  • This rapid and affordable sequencing approach is valuable for selecting improved protein variants, particularly when activity is linked to growth.