# A cost-effective and scalable barcoded library construction method for deep mutational scanning studies

## TL;DR

This paper introduces a cost-effective and scalable method for building DNA variant libraries, enabling efficient deep mutational scanning for large genes.

## Contribution

The novel approach uses low-cost DNA synthesis and multiple barcodes per variant to eliminate the need for long-read sequencing.

## Key findings

- The method was successfully applied to construct a complete library for the 3.2 kb PDR1 gene in yeast.
- Barcode sequencing showed near-perfect agreement with direct sequencing of mutated coding sequences.
- The approach is scalable and reduces the technical and financial barriers for studying large genes.

## Abstract

Recent developments in DNA synthesis and sequencing allow the construction of comprehensive gene variant libraries and their functional analysis. Achieving high-replication and thorough mutation characterization remains technically and financially challenging for long genes. Here, we developed an efficient, affordable, and scalable library construction approach that relies on low-cost DNA synthesis and standard cloning technologies, which will increase accessibility to mutational studies and help advance the field of protein science. Each degenerate codon variant is physically associated with multiple DNA barcodes during synthesis, which overcomes the need for long-read sequencing for linking variants to barcodes. We demonstrate the scalability of our approach by constructing a complete library for the multidrug resistance gene PDR1, a 3.2 kb gene encoding a pleiotropic transcription factor in the yeast Saccharomyces cerevisiae. We demonstrate a near-perfect correspondence in the measurement of amino acid variants impact when assessed by barcode sequencing and direct sequencing of the mutated coding sequence.

The scalability of deep mutational scanning (DMS) experiments are limited by gene size due to the complexity of the variant library and the costs of DNA synthesis and sequencing. This study develops an efficient and cost-effective barcoded cloning strategy for plasmid-based DMS libraries that facilitates the study of large genes.