Scaling logical density of DNA storage with enzymatically-ligated composite motifs.

DNA is a promising candidate for long-term data storage due to its high density and endurance. The key challenge in DNA storage today is the cost of synthesis. In this work, we propose composite motifs, a framework that uses a mixture of prefabricated motifs as building blocks to reduce synthesis cost by scaling logical density. To write data, we introduce Bridge Oligonucleotide Assembly, an enzymatic ligation technique for synthesizing oligos based on composite motifs. To sequence data, we introduce Direct Oligonucleotide Sequencing, a nanopore-based technique to sequence short oligos, eliminating common preparatory steps like DNA assembly, amplification and end-prep. To decode data, we introduce Motif-Search, a novel consensus caller that provides accurate reconstruction despite synthesis and sequencing errors. Using the proposed methods, we present an end-to-end experiment where we store the text "HelloWorld" at a logical density of 84 bits/cycle (14-42× improvement over state-of-the-art).

Direct oligonucleotide sequencing with nanopores.

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