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

NPlasmid-seq uses Cas9-guided paired terminal signatures to detect sub-percent plasmid contamination, achieving precision 1.000 with no false positives across 493,200 target reads.

• Why it matters: Consensus plasmid QC can miss structural heterogeneity and low-level contaminants, limiting confidence in vector and IVT-template preparations.
• What they did: The workflow cleavage-validated 24 sgRNAs and applied a class-specific contaminant classifier to computational and physical DNA mixtures, plus read-level analysis of structural variants.
• The result: In computational 5,000-read subsamples, contaminants were detected at 0.2–5% in all 20 subsamples and at 0.1% in 13/20, while physical mixtures detected contaminants at 0.5 and 1 mol% and read-level analysis revealed a recurrent 93-bp deletion.

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