Fig. 4: Demonstration of digital music data storage in DNA oligonucleotides enzymatically synthesized in multiplex: music data storage process.

a A snapshot of the simplified melody from the two first measures of the 1985 Nintendo Entertainment System video game Super Mario Brothers “Overworld Theme” piano sheet music. Each note and rest of the melody was indexed as #0 through #11 and stored into one of the 12 unique DNA oligonucleotides synthesized in multiplex on the (3 ⋅ 4) array. The full piano sheet music is shown in Supplementary Fig. 7. b Indexed notes and rests were assigned a note number based on a modified Musical Instrument Digital Information note chart, which also indicates note octave. In addition to the note, the encoding scheme also stores the order and duration each note is to be played. All rests are assigned to the note G#₀, which is inaudible (25.9 Hz) to normal human hearing²⁵. A full overview of MIDI conversion of sheet music to digital data is shown in Supplementary Fig. 8. c Digital music data is translated to ternary and the unique DNA sequences are mapped to unique base transitions using a conversion map. The left axis of the table represents the string of bases at the 3′- terminus of the initiator oligonucleotide and the top axis indicates the nucleotide to be incorporated next. Supplementary Figure 9 indicates a table outlining the mapped “Overworld Theme” musical melody to DNA sequences with all relevant data and information. d Generated DNA sequences are enzymatically synthesized with TdT using our multiplex photolithographic system. e Synthesized oligonucleotides are retrieved from the flow cell surface and stored in tubes for sequencing or other downstream applications. f To read and decode stored digital music data, DNA oligonucleotides synthesized in multiplex can be sequenced with NGS such as Illumina or nanopore methodologies. g From sequencing data, the decoding process converts sequencing information back to musical notes. A sinusoidal wave generator is used to play the “Overworld Theme” melody in the correct note order with the proper duration in true sound.