CUBED: Universal combinatorial barcoding platform for highly multiplexed digital PCR.

Highly multiplexed molecular assays are essential for applications ranging from pathogen detection to genetic and copy number analysis. Here, we introduce CUBED (Combinatorial Universal Barcode End-point Digital PCR), a platform that enables simultaneous detection and quantification of numerous nucleic acid targets in a single reaction using standard endpoint digital PCR instruments. CUBED combines combinatorial color barcoding with multi-amplitude probe labeling to expand multiplexing capacity beyond the limits imposed by fluorescence channel count. An integrated error-detection scheme minimizes misclassification from signal dropout, ensuring robust decoding of complex barcodes. We demonstrate accurate quantitative detection and discrimination of 26 ternary barcodes using only three fluorescence channels, and separately resolve 20 binary barcodes on a six-channel system. We further apply CUBED to pooled SARS-CoV-2 RNA detection across 20 uniquely barcoded samples, confirming its quantitative accuracy and scalability. This framework provides a high-throughput, error-tolerant solution for multiplexed nucleic acid detection in research and clinical diagnostics.