Fig. 1

Schematic overview of the dual-barcoded shotgun expression library sequencing (Dub-seq) approach. a A pair of random 20 nucleotide DNA sequences, the UP and DOWN (DN) barcodes are cloned into an expression vector. Deep sequencing of the dual-barcoded vector (BPseq) associates UP and DOWN barcode sequences. b Target genomic DNA is randomly sheared and cloned between the UP and DOWN barcodes to create the Dub-seq plasmid library. c To characterize the Dub-seq library, a “Tn-seq” like protocol is performed to precisely map the two genomic breakpoints of each insert and to associate each breakpoint with its random DNA barcode sequence. If the source genome(s) has been sequenced, then BAGseq can be used to define the exact sequence of each plasmid in the library. d The fitness of bacteria carrying different plasmids can be measured with pooled growth assays and deep sequencing of the DNA barcodes (BarSeq). Strain (or fragment) fitness is defined as the log₂ ratio of barcode abundance after selection (end) versus before (start). Gene fitness is estimated from the fragments’ fitness by a constrained regression