NinjaSeq: programmable restriction enzyme-based sequencing library preparation with random access for DNA data storage
Year: 2026
Type: preprint
Abstract: SUMMARY DNA data storage allows sequences to be defined without biological constraints, yet readout workflows still depend on generic end-repair/dA-tailing chemistry. We developed NinjaSeq, a type IIS restriction endonuclease library-preparation strategy that incorporates recognition sites into primer flanks, enabling digestion to generate adapter-compatible overhangs and eliminating the need for conventional end preparation. By combining this chemistry with constrained coding that excludes internal recognition motifs, NinjaSeq produced sequencing quality and decoding performance consistent with standard protocols while reducing reagent burden and simplifying processing, including compatibility with one-pot restriction-ligation. The same sequence-directed design also enables physical random access during library preparation: targeting file-specific flanking sites enriched a desired file from a mixed pool by about sixteen-fold in a proof-of-concept experiment. These results position NinjaSeq as a practical ONT readout approach for DNA data storage. HIGHLIGHTS NinjaSeq replaces end-repair/dA-tailing with REases for nanopore sequencing Constrained encoding excludes recognition motifs to protect payloads from cleavage NinjaSeq achieves decoding accuracy comparable to standard library preparation Designing file-specific RRS enables random access during library preparation
Language: English
Topic: DNA and Biological Computing
Subfield: Theoretical Computer Science
Field: Mathematics
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7164586702