Abstract

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in C. elegans, E. coli, S. cervisiae, and C. reinhardtii. This method, demands merely 20 minutes of hands-on time for a single worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the oncogenic BRAF(V600E) mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species.