Robust higher-order multiplexing in digital PCR by color-combination

There is a growing need in molecular biology to interrogate samples for higher number of biomarkers, beyond the 2 to 5 biomarkers typically addressable with standard PCR technologies. Here, we demonstrate a novel approach to increase the level of multiplexing in digital PCR up to 15-plex by detecting each target with 2 distinct fluorophores with a 6-color digital PCR system, a method called digital PCR by color combination. We provide a statistical framework to interpret digital PCR data by color combination, predicting that high-plexed assays by color combination can, in theory, have the same precision and sensitivity as corresponding single-plex assays. We verified the statistical framework and confirmed the sensitivity prediction experimentally on a 15-plex assay and an 11-plex assay. These results establish the technical framework for future high-plex assays addressing a variety of applications and utilities, such as infectious disease syndromic panels or tumor mutation profiling panels.