Direct oligonucleotide sequencing with nanopores

Third-generation DNA sequencing has enabled of long, unamplified fragments with minimal steps. Direct ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add complimentary strand. single-stranded nucleic acid species is challenging as they are non-compatible double-stranded adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs passing single-strands through a layer biological nanopore sensors; although performed on single-strands, recommended template manufacturer double-stranded. We have identified that can perform short, single-strand oligonucleotides directly amplification second-strand performing single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed an adapter sequence be sequenced in 5' 3' direction via nanopores. Adapter sequences were designed bind end leave 3’ adenosine overhang after binding their target. terminal phosphate makes oligo analogous end-prepared dsDNA, rendering it compatible ligation-based preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal was using this method ~90% these recovered high accuracy BLAST. In raw data, we empty signals wrongly valid read sometimes multiple several strands fused into file due segmentation faults software. This direct oligonucleotide enables novel applications data storage systems where short primary information carriers.