Protocol for the preparation of a high quality transposon DNA library for NGS. (A) (1) Genomic DNA is isolated and digested with NotI. High molecular weight DNA is selectively precipitated using an 8%PEG + NaCl solution, and transposon-plasmid junctions (106 bp) are removed in the supernatant. A biotinylated dsDNA adapter with NotI overhang is ligated (2) before digestion with MmeI, which cuts non-specifically 20 bp from its recognition site within ITR2 into the genome to liberate biotinylated-transposon-genome junctions as short DNA fragments (114 bp). (3). Biotinylated fragments are bound to streptavidin beads (4), and an Illumina sequencing primer adapter containing an indexing barcode and MmeI compatible ends is ligated (5). Primers annealing to the P7 site and the Illumina sequencing primer adapter sequence (with a P5 site overhang) are used to PCR amplify the transposon-genome junctions (6), agarose gel purified, and submitted for Illumina sequencing (7). NGS reads capture both the 16-bp of flanking genomic DNA as well as the transposon donor specific barcode located between the P7 and ITR2. (B) Fragments arising from transposon-plasmid junctions are removed by size selective PEG-NaCl precipitation, while the remaining fragments lack both P7 annealing sites and MmeI sites for ligation of the Illumina sequencing primer adapter. These fragments are therefore not amplified in step (6) of 4A. (C) By performing the size-selective precipitation on a 1 kb DNA ladder, we show that small 300 bp fragments of DNA are retained in the solution (SN), while larger DNA is precipitated (P). (D) Six transposon donor constructs were multiplexed and designed to attenuate expression of genes proximal to the insertion site according to the regulatory elements located at the ends of the transposon backbone. Each donor can be identified from NGS reads by the unique 3 bp barcode.