Bioinformatics Evidence

 Quick Analysis Plan Copied

 Long Analysis Plan

Plan: tiered confidence framework for blaNDM-5 IS26-composite transposon inference

Step 1 — Evidence baseline. The population-scale observation is that blaNDM-5 occurs consistently in an IS26-flanked context adjacent to a class 1 integron in ST405, with 29 genomes providing intact 20 kb flanks and 564 providing only partial flanks for context comparison via CD-HIT clustering and maximum-likelihood context phylogeny Global ST405 blaNDM-5 IS26 context survey. Mechanistic grounding comes from the IS6 family review: IS26 is 789 bp with 15–20 bp inverted repeats, generates 8 bp direct target repeats on insertion, carries promoter elements that can drive neighboring gene expression, and mobilizes resistance genes via cointegration, tandem amplification, and circular translocatable units IS6 family mechanism review.

Step 2 — Computational workflow. (a) Retrieve complete plasmid sequences (long-read/hybrid assemblies only); (b) extract ±20 kb around blaNDM-5; (c) detect IS26 copies (ISfinder BLAST; expect the 789 bp element and 8 bp DRs); (d) confirm both flanking IS26 copies with correct orientation and intact IRs; (e) cluster contexts (CD-HIT) against the known NDM-5 context clusters; (f) assign confidence tiers: High = closed plasmid, two intact IS26 ends, 8 bp DRs, context matching a known cluster; Moderate = complete flanks on a closed genome but atypical arrangement; Low = draft assembly or one partial flank — analogous to the 564 partial-flank genomes, which should not be called composite transposons without long-read support Cross-ST plasmid sharing evidence.

Step 3 — Validation anchors. IS26 copy number varies widely even within one plasmid system (8, 4, and 3 copies on the K. pneumoniae pTGC plasmids), so count alone is not diagnostic; IS26-mediated homologous recombination generates hybrid plasmids detectable by comparing transconjugant vs parental plasmid maps IS26-mediated plasmid rearrangement counts. Experimental validation via RecA-dependence shows that tandem amplification and circular TU formation are largely RecA-dependent homologous recombination events, not solely transposase-driven, meaning genomic adjacency alone does not prove recent composite-transposon mobilization RecA-dependent IS26 TU amplification.

Step 4 — Blind spots. TU intermediacy rests mainly on one well-studied case and lacks general biochemical proof TU intermediacy caution. Disagreement also exists over whether targeted conservative cointegration extends beyond IS26 within the IS6 family, a live dispute that affects how confidently composite-transposon labels generalize IS26 mechanism scope dispute. Falsifiers: a blaNDM-5 context lacking both IS26 ends on a closed plasmid; DR sequences inconsistent with IS26 insertion; or demonstration that flanking IS26 copies predate blaNDM-5 acquisition.