De novo non-canonical nanopore basecalling enables private communication using heavily-modified DNA data at single-molecule level.

Hidden messages in DNA molecules by employing chemical modifications has been suggested for private data storage and transmission at high information density. However, rapidly decoding these "molecular keys" with corresponding basecallers remains challenging. We present DeepSME, a nanopore sequencing and deep-learning based framework towards single-molecule encryption, demonstrated by using 5-hydroxymethylcytosine (5hmC) substitution for individual nucleotide recognition rather than sequential interactions. This non-natural, motif-insensitive methylation disrupts ion current, resulting in a readout failure of 67.2%-100%, concealing the privacy within the DNAs. We further develop an alignment-free DeepSME basecaller as a key to reconstitute the digital information. Our three-stage training pipeline, expands k-mer size from 4 to 4, achieving over 92% precision and recall from scratch. DeepSME deciphers fully 5hmC concealed text and image within 16× coverage depth with an F1-score of 86.4%, surpassing all the state-of-the-art basecallers. Demonstrated on edge computing devices, DeepSME holds supreme potential for DNA-based private communications and broader bioengineering and medical applications.