Reduction of transposon-plasmid junction NGS reads with flanking
I restriction sites. (A) Inverse PCR was used to amplify the ITR2 transposon junctions for twelve colonies as has been described . Three out of twelve of these colonies also contained transposon-plasmid junctions (~160 bp DNA band). This ratio increased to seven out of twelve when the canonical ITR sequence was altered to incorporate a MmeI recognition site (Additional file 1: Figure S2). Results are representative of multiple independent experimental replicates. (B) The putative mechanism for transposase catalyzed integration of transposon-plasmid junctions may involve engagement of non-contiguous ITR repeats (dashed lines), resulting in chromosomally integrated transposon multimers. In contrast, when both ITR sequences are optimal, contiguous ITRs are most frequently mobilized (solid lines). (C) Colors are used to identify the positions of the sequences in this drawing. To selectively remove transposon-plasmid junctions, we introduced two NotI sites into the transposon construct that flanked the MmeI modified ITR2. In addition, we included a P7 Illumina sequencing primer site with a unique 3-bp DNA barcode to identify the P
out promoter that faces outward from ITR1 during NGS sequencing. (D) After first digesting gDNA with NotI, the transposon-plasmid junction content was substantially reduced in comparison to Figure 3A.