Detection of low-frequency mutant alleles in serum-derived, circulating, cell-free DNA (ccfDNA) from cancer patients using multiplexed preamplification in digital PCR. Water alone [no template control (NTC)], wild-type ccfDNA, and ccfDNA from three cancer patients with the KRAS (patient MS1079; A), TP53 (patient MS310; B), or SMAD4 (patient 4873; C) mutation were subjected to multiplexed preamplification and assessed for presence of the mutation using digital PCR. A single digital PCR was performed on each patient ccfDNA sample to assess presence of mutation without preamplification (right panels). The numbers shown in the bottom right of each plot indicate the number of mutant alleles detected for each condition. P values are denoted in the top right of each plot. Arb., arbitrary.