uSort-M: Scalable isolation of user-defined sequences from diverse pooled libraries

Area of Science:

• Biochemistry and Molecular Biology
• Genomics and Proteomics
• Synthetic Biology

Background:

• High-throughput sequencing and computational protein design accelerate protein discovery but outpace functional characterization.
• Current methods for individual variant analysis are limited by the high cost of gene synthesis, restricting studies to small variant subsets.
• A significant gap exists between identifying novel protein sequences and understanding their functions due to analysis bottlenecks.

Purpose of the Study:

• To develop a cost-effective and efficient workflow for isolating and identifying individual protein variants from large, pooled libraries.
• To overcome the limitations imposed by expensive gene synthesis in large-scale functional protein characterization.
• To enable rapid functional annotation of diverse protein variants discovered through high-throughput methods.

Main Methods:

• Developed user-defined Sorted Mutants (uSort-M), integrating pooled DNA synthesis, automated cell sorting of *Escherichia coli*, and long-read sequencing.
• uSort-M accommodates pooled libraries generated by various methods, including multiplex assembly, error-prone PCR, and pooled site-directed mutagenesis.
• Automated sorting of single bacterial clones into 384-well plates achieved high efficiency (up to 90% monoclonal cultures) within 1-2 hours for eight plates.

Main Results:

• Successfully isolated and identified individual variants from pooled libraries using uSort-M.
• Application to a 328-member scanning mutagenesis library recovered 96% of desired variants at fivefold lower cost than traditional synthesis.
• Long-read sequencing provided accessible, fast, and cost-effective identification of sequences, tolerating library diversity and fragment length variations.
• Numerical simulations demonstrated the workflow's scalability and predicted sampling effort for target coverage.

Conclusions:

• uSort-M significantly reduces the cost and time required for isolating and sequencing individual protein variants from pooled libraries.
• The workflow's generalizability, efficiency, and use of standard instrumentation remove a key barrier to large-scale protein functional characterization.
• This method facilitates bridging the gap between protein discovery and functional annotation, accelerating biological research.