Direct oligonucleotide sequencing with nanopores

Abstract Third-generation DNA sequencing has enabled users to sequence long, unamplified DNA fragments with minimal sample and library preparation steps. Sequencing single-stranded nucleic acids directly without amplification or by ligating a spacer strand are challenging, as the single-strand species are poor templates to add the sequencing adapters. Sequencing ssDNA or RNA directly gives valuable insights like base-level modifications and degradation levels along with saving valuable time and resources. Biological nanopores used by Oxford Nanopore Technologies process the target strands at a single-strand level, although the typical samples sequenced are double-stranded or converted into double-strand. We have identified that the MinION platform from Oxford Nanopore can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing an annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores, the adapters were designed to bind to the 5’ end of the oligos and leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo to be analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000 orthogonal sequences of 120 bp length were sequenced using the method and 37,265 of the total sequences were recovered with high accuracy. While analyzing the raw data, we had interesting observations. In our raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single read by the platforms segmentation faults. We believe that this method could enable novel applications of nanopore sequencing in DNA data-storage systems where short oligonucleotides function as the primary information carriers.