Comparison of library uniformity (TP0) between two DMS plasmid library construction strategies.

This table presents a quantitative comparison of the variant uniformity achieved by our scalable barcoded strategy PDR1 DMS TP0 F13 library and a reference DMS plasmid library CaERG11 previously generated in our laboratory [5]. The CaERG11 library was constructed using a highly precise, low-throughput method: DNA fragments synthesized by Twist Bioscience were cloned codon-by-codon into a yeast expression vector, and the individual codon libraries were then pooled together proportionally based on the number of mutants. This meticulous process allows to achieve a near-perfect uniformity of variant representation, which serves here as a gold-standard reference. The comparison indicates that the uniformity achieved by our barcoded strategy is certainly less efficient than the CaERG11 reference, but it is perfectly acceptable for robust DMS experiments (Gini coefficient around 0.52). Crucially, the data demonstrate that our method achieves this acceptable uniformity while remaining substantially more cost-effective and scalable. The metrics computed include the Gini coefficient (0 = perfect uniformity, 1 = maximum bias) and the Uniformity Score. (XLSX)