Restriction-site-based enrichment coupled to adaptive sampling enables long-read transposon-insertion sequencing

Long-read Transposon insertion sequencing is essential for linking genotypes to phenotypes, as short- read approaches struggle with complex genomic regions. Long-read sequencing demands high sequencing depth or targeted sequencing, which either requires extensive sample preparation or suffer from low net efficiency. We developed a simple, low-cost workflow combining enzymatic cleavage with adaptive sampling to drastically improve target recovery and simplify analysis. I-SceI restriction site was introduced into a mini-Tn5 transposon to generate a mutant library in Cupriavidus necator. Prior to Nanopore sequencing, gDNA was digested to introduce double-strand breaks precisely at insertion sites, followed by sequencing with adaptive sampling. The workflow was validated via a biofilm selection screen. Combining I-SceI digestion with adaptive sampling yielded an effective 11.3-fold enrichment (after normalizing for pore occupancy). High-depth sequencing covered 97.1% of all genes. Because reads consistently began at the exact insertion site, bioinformatic trimming was bypassed. The biofilm screen successfully identified enriched integration loci, uniquely mapped insertions within repetitive rRNA operons, and revealed co-selected, spontaneous secondary mutations.This method adds less than one hour to library preparation, reduces consumable costs by over 90%, and establishes a budget-friendly, simple and high-resolution long-read Tn-seq pipeline ideal for complex and multipartite genomes.