Nanopore-Based Glycan Sequencing via a Fragmentation-Reassembly Strategy

Area of Science:

• Biotechnology
• Analytical Chemistry
• Glycoscience

Background:

• Nanopore sequencing offers real-time oligosaccharide analysis.
• Previous work proposed hydrolysis, strand, and assembly sequencing strategies.
• The feasibility of reconstructing full glycan sequences from fragments was unestablished.

Purpose of the Study:

• To evaluate the nanopore-based fragmentation-reassembly strategy for glycan sequencing.
• To establish the feasibility of reconstructing complex glycan structures from fragmented data.
• To validate a modular nanopore-based framework for glycan decoding.

Main Methods:

• Complex glycans were hydrolyzed into fragments.
• Fragments were detected using a mutant nanopore and classified.
• Set-theoretic operations integrated predictions to reconstruct glycan structures.

Main Results:

• Achieved 93.71% reconstruction fidelity for branched N-glycans.
• Demonstrated robustness against similar glycans, complexity, and biological background.
• Successfully reconstructed the branch structure of a model N-glycan.

Conclusions:

• The fragmentation-reassembly strategy is a viable method for nanopore-based glycan sequencing.
• This approach enables structural inference from partial fragment information, reducing effort.
• Represents the first demonstration of single-molecule sequencing for branched glycans using nanopores.