Coupling filter mating with nanopore selective sequencing expands capturable conjugative plasmid recovery in biological wastewater treatment systems

Wastewater treatment plants (WWTPs) are hotspots for the exchange of plasmids, which can spread antibiotic resistance genes. However, recovering conjugative plasmids from complex communities remains challenging because both plasmid extraction and cultivation-based approaches introduce bias. Here we show that filter mating coupled with nanopore selective sequencing (FM-NSS) can recover an ampicillin-selected, recipient-compatible fraction of conjugative plasmids from a full-scale WWTP without plasmid extraction. After depletion of reads aligning to the Escherichia coli recipient genome, FM-NSS recovered a total of 1.10 million plasmid reads. De novo assembly yielded 622 non-redundant plasmid contigs, including 441 not detected by extraction-based plasmid sequencing from the same conjugant pools. Notably, activated sludge displayed a distinct recovered plasmid profile compared with other treatment stages, which is associated with an elevated estimation of community replication rates. FM-NSS provides a practical workflow to expand the recovery of selected conjugative plasmids from complex environmental communities. © The Author(s), under exclusive licence to Springer Nature Limited 2026.