Strategy for using phage-based transposition to make high quality transposon libraries for NGS sequencing. (A) The transposon insertion library is made by creating a high-frequency transducing lysate of the transposon cassette that is able to replicate as a plasmid in the donor strain (repC
+). The lysate is mixed with the recipient strain (repC
−) carrying a temperature sensitive plasmid from which the Himar1 transposase is expressed, and erythromycin resistant transposon insertion mutants are selected. (B) By fitting the transposon cassette with an outward-facing promoter, genes can be up- or down-regulated, or inactivated if non-essential, in a single library pool. In order to cover a wide range of gene expression levels, different promoter containing transposon constructs can be multiplexed and then identified during NGS sequencing by unique DNA barcodes (purple bar) to collect fitness-gene dose relationships by monitoring read counts.