Expanding the Molecular Alphabet of DNA-Based Data Storage Systems with Neural Network Nanopore Readout Processing

[Image: see text] DNA is a promising next-generation data storage medium, but challenges remain with synthesis costs and recording latency. Here, we describe a prototype of a DNA data storage system that uses an extended molecular alphabet combining natural and chemically modified nucleotides. Our results show that MspA nanopores can discriminate different combinations and ordered sequences of natural and chemically modified nucleotides in custom-designed oligomers. We further demonstrate single-molecule sequencing of the extended alphabet using a neural network architecture that classifies raw current signals generated by Oxford Nanopore sequencers with an average accuracy exceeding 60% (39× larger than random guessing). Molecular dynamics simulations show that the majority of modified nucleotides lead to only minor perturbations of the DNA double helix. Overall, the extended molecular alphabet may potentially offer a nearly 2-fold increase in storage density and potentially the same order of reduction in the recording latency, thereby enabling new implementations of molecular recorders.