NPlasmid-seq: Cas9-encoded terminal signatures enable molecule-level nanopore quality control of vector and IVT-template plasmids

Consensus-based plasmid quality control can overlook structural heterogeneity within a preparation. We developed NPlasmid-seq, a Cas9-guided nanopore workflow that encodes sample identity through paired terminal signatures generated at predefined backbone positions, without sample-specific oligonucleotide barcodes. We cleavage-validated 24 sgRNAs. In five representative assigned read bins, each bin showed its strongest sequence agreement with its cognate reference. Contaminant detection was evaluated with a single class-specific classifier applied both to computational read mixtures and to physical DNA mixtures, placing the two designs on a common scale. In computational read mixtures of 5,000 reads, a contaminant was detected in all 20 independent subsamples at each of 0.2%, 0.5%, 1% and 5% abundance and in 13 of 20 subsamples at 0.1%; precision was 1.000 in all 100 subsamples, with no false-positive calls among 493,200 reads from the declared target plasmid. In physical DNA mixtures carried through cleavage, pooled library preparation and sequencing, a contaminant was detected in all six samples prepared at 0.5 mol% input and all six at 1 mol%. Observed read fractions did not distinguish the 0.5 and 1 mol% input levels. Instead, they differed systematically by 4.0-fold between preparation pools, a larger effect than the difference between the two input levels. We therefore interpret the workflow as a detector, not a quantifier, of sub-percent contamination. Read-level analysis identified a recurrent 93-bp deletion in 61.7% of reads from a preparation that had passed consensus-based quality control. Sanger genotyping of 24 colonies identified deletion-type profiles in 10 colonies and intact-type profiles in three. The remaining 11 colonies showed double peaks at the diagnostic locus, consistent with coexistence of both sequence forms within individual clones. In in vitro transcription template plasmids, longer poly(A) tracts were associated with higher multimeric-read fractions. Across ten matched A90 culture pairs, propagation at 30 degree reduced these fractions relative to 37 degree (paired t-test, P = 0.0098). These measurements describe recovered read populations, not absolute molecular abundances. NPlasmid-seq therefore complements consensus validation by revealing sequence and structural heterogeneity within a preparation and by identifying a culture condition associated with a lower multimeric-read fraction.