Dibenzocyclooctyne-modified PCR primers enable direct enzyme-free click chemistry ligation for custom nanopore amplicon sequencing

Oxford Nanopore Technologies (ONT) rapid library preparation kits use transposase-mediated tagmentation to attach click chemistry functionalized oligonucleotide duplexes to fragmented DNA, followed by click chemistry to conjugate Rapid Adapter (RA) sequencing adapters. A similar protocol is used in 16S rRNA gene amplicon and PCR-amplified rapid whole-genome sequencing workflows. Here, we describe custom oligonucleotides with dibenzocyclooctyne (DBCO) added onto PCR primer 5' termini. After standard PCR amplification, DBCO-modified amplicons react spontaneously with RA sequencing adapters, producing sequencing-ready libraries in minutes without enzymatic processing. All configurations employ an asymmetric design in which the DBCO modification is restricted to a single primer, leaving the opposite primer available for barcoding at low cost. We validate three primer architectures: (i) direct attachment of DBCO to a target-specific primer, (ii) a universal DBCO-modified oligonucleotide used in a two-step PCR workflow, and (iii) a three-primer single-pot reaction combining the universal DBCO oligonucleotide with unmodified target-specific primers. These configurations are validated using full-length 16S rRNA gene amplicons sequenced on a PromethION flow cell. DBCO-modified primers are synthesized either commercially or in-house via DBCO-TFP ester conjugation to 5'-amino oligonucleotides and remain fully active through standard PCR thermocycling. The best-performing configuration used a two-step PCR with a universal oligonucleotide and achieved higher pore occupation and reads than comparable commercial solutions. This approach reduces library preparation reagent costs compared to available kits, as the initial synthesis cost is lower than existing amplicon sequencing kits, while providing enough material for hundreds or thousands of PCR reactions. This is further applicable to an unlimited number of gene targets beyond 16S sequencing.