Digital Droplet PCR (ddPCR) for Absolute Quantification of 16S rRNA Copy Number in Metagenomic Data

Digital Droplet PCR (ddPCR) is a quantitative PCR method that offers high sensitivity and accuracy in measuring the amount of nucleic acid in a sample, without the need of a standard curve. In ddPCR, a single sample is partitioned into up to 20,000 droplets, using the water–oil emulsion technology, and the amplification reaction occurs within each droplet using a fluorescent hydrolysis probe (Taqman) or a DNA-binding fluorescent dye. Following PCR, the emitted signals are individually measured in each droplet. Here, we describe a ddPCR optimized protocol for accurately quantifying the total copy number of the 16S rRNA gene in a metagenomic DNA sample. The protocol utilizes a primer pair, targeting the 16S V5-V6 hypervariable regions, in combination with a double-strand DNA-binding fluorescent dye. © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2026.