Design and Assembly of Combinatorial DNA Barcodes for Probe-based Genomics Applications [Pyxa spatial transcriptomic]

Here we present a modular framework that integrates custom software tools with purposebuilt cloning reagents to design, assemble, validate, and deploy "combinatorial" DNA barcodes. 25 Combinatorial barcodes comprise spatially adjacent collections of known sequences, enabling millions of unique molecules to be efficiently distinguished using a limited set of probes.

Our software tools integrate with optimized assembly plasmids and whole plasmid long-read sequencing for highfidelity construction and structural validation of diverse combinatorial barcode architectures. Assembled barcode libraries are flexibly transferred into user-modified expression vectors to support 30 diverse downstream experimental applications. We showcase the versatility of this framework by assembling two structurally distinct combinatorial barcode libraries, each containing millions of unique sequences.