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A major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that uses random DNA barcodes to greatly increase experimental throughput. As a demonstration of this approach, we construct a Dub-seq library with  Escherichia coli  genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified overexpression phenotypes for 813 genes. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for  E. coli  genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches and will facilitate rapid and systematic functional characterization of microbial genomes.

Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria