A practical DNA data storage using an expanded alphabet introducing 5-methylcytosine

The DNA molecule is a promising next-generation data storage medium. Recently, it has been theoretically proposed that non-natural or modified bases can serve as extra molecular letters to increase the information density. However, this strategy is challenging due to the difficulty in synthesizing non-natural DNA sequences and their complex structure. Here, we described a practical DNA data storage transcoding scheme named R+ based on an expanded molecular alphabet that introduces 5-methylcytosine (5mC). We demonstrated its experimental validation by encoding one representative file into several 1.3∼1.6 kbps in vitro DNA fragments for nanopore sequencing. Our results show an average data recovery rate of 98.97% and 86.91% with and without reference, respectively. Our work validates the practicability of 5mC in DNA storage systems, with a potentially wide range of applications. Availability and implementation R+ is implemented in Python and the code is available under a MIT license at https://github.com/Incpink-Liu/DNA-storage-R_plus.