Restriction-site-based enrichment coupled to adaptive sampling enables long-read transposon-insertion sequencing
Abstract
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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/733934v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@110f3eborg.highwire.dtl.DTLVardef@1fdcdf3org.highwire.dtl.DTLVardef@1dc293corg.highwire.dtl.DTLVardef@1175f9e_HPS_FORMAT_FIGEXP M_FIG C_FIG
Full-text reasoning
From the deep-tier full-text analysis of this preprint.
Reasoning review — 6 key claims, 0 well-supported, 6 with gaps
- partial The new method is simple, budget-friendly, high-resolution, adds <1hr to prep, and reduces consumable costs >90%. gap: The text asserts significant time and cost reductions and high resolution without presenting any comparative data or quantitative evidence.
- unsupported Our method improves target enrichment by >10x when combined with adaptive sampling by introducing an additional restriction site. gap: The text states a quantitative enrichment improvement (>10x) but provides no data, figures, or statistical analysis to substantiate this claim.
- unsupported The method simplifies analysis workflow and has DNA input demand on par with standard sequencing protocols. gap: The text claims simplification of analysis and comparable DNA input without presenting any evidence or comparative data.
- unsupported A biofilm selection experiment illustrates the method's applicability for phenotype-driven enrichment in complex, multipartite genomes. gap: The text refers to an experiment and its illustrative power but provides no details of the experiment or its results to demonstrate applicability.
- unsupported The method demonstrates advantages of native long reads over PCR-based and short reads in TIS regarding sensitivity, specificity, and statistical analysis. gap: The text asserts advantages of long reads and their method over short reads without presenting any comparative data or analysis to support these claims.
- unsupported The method is extremely easy to apply, resulting in greater accessibility and reduced costs compared to Cas9-based approaches. gap: The text claims ease of application and cost reduction compared to other methods without providing any comparative data or evidence.
Claims and gaps are read from the full text by a language model, shown for transparency; they do not affect ranking or selection.
Lifecycle
- biorxiv v1 2026-06-28 source ↗
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