Nanopore-Based Glycan Sequencing via a Fragmentation-Reassembly Strategy.

Nanopore have emerged as a promising platform for glycan sequencing, enabling the real-time readout of individual oligosaccharides. Previously, we proposed three conceptual nanopore-based strategies for glycan decoding including hydrolysis sequencing, strand sequencing, and assembly sequencing. However, the feasibility of reconstructing the full sequence of a glycan from fragments has not yet been established. Here, we performed a proof-of-concept evaluation of the nanopore-based fragmentation-reassembly strategy. Intact complex glycans were hydrolyzed into fragments, which were detected by a nanopore mutant and assigned to structural candidates using a trained classifier. The resulting predictions were integrated through set-theoretic operations, where intersections identified shared structural elements, unions incorporated branch-specific features, and set differences removed incompatible combinations. Through these fragment-integration steps, the branch structure of the model N-glycan was reconstructed. Under this sequencing system, we achieved 93.71% reconstruction fidelity for branched glycans, and the approach remained robust in the presence of structurally similar glycans, compositional complexity, and biological background, highlighting its potential for real-world applications. The sequencing workflow could be broadly applicable across glycan types and adaptable to different nanopore systems. Unlike approaches requiring exhaustive fragment coverage, this strategy showed that coherent structural inference may be possible from partial fragment information while reducing measurement effort and database demands. To the best of our knowledge, this represents the first demonstration of nanopore-based single-molecule sequencing of branched glycans. Together with prior demonstrations of hydrolysis and strand strategies, this work completes the initial validation of a modular nanopore-based framework for glycan decoding.