Fig. 3: Demonstration of multi-cycle and multiplex enzymatic synthesis.

a An overview of multi-cycle, single-plex synthesis of a heteropolymer oligonucleotide comprised of 8 unique base transitions. Each cycle represents the addition of a single-nucleotide base type at all 12 spots on the (3 ⋅ 4) array. Synthesis results in short homopolymeric blocks of A, T, G, or C at variable lengths. b Following multi-cycle synthesis and NGS, raw sequence reads were extracted and filtered by the presence of the adapter sequence added by splint-end ligation after the final C extension. The box-plot indicates a statistical representation of the number of extension events for each homopolymeric block for the synthesized sequence GATGTAGAC with red pluses being statistical outliers. Note that only sequencing reads that contained all eight base transitions were used to generate the box-plot and that synthesis started with a string of four G on the 3^′-terminus of the initiator oligonucleotide. c Denaturing gel electrophoresis analysis of each individual cycle of synthesis. Each lane represents the material retrieved of an individual flow cell after the appropriate number of cycles were performed. No final C extension or splint-end ligation occurred for this analysis. d An overview of multi-cycle, multiplex synthesis of 12 unique heteropolymer oligonucleotides on the (3 ⋅ 4) array. Each synthesis step contained the individual cycles for the addition A, T, G, and C at the appropriate spots on the array. Sequence barcodes indicating the physical location of each oligonucleotide on the array can be built into the initiator oligonucleotide or synthesized by TdT. For each cycle, a unique mask was generated by the system’s DMD to locally activate TdT based on the desired sequence to be synthesized. e For example, the masks needed for the 4th step of an eight-step multiplex oligonucleotide synthesis run is shown with the appropriate post-synthesis sequencing data to verify spatially selective synthesis. Source data are provided as a Source Data file.