Detection of low levels of mutations by LCN-HRM and characterisation by subsequent sequencing. HCT116 and NCI-H1650 cell line DNA were mixed with normal DNA to a 5% mutant allele frequency based on the previous qPCR data. Each of the DNA mixtures were then tested by LCN-HRM in 65 replicates. An estimated average of three copies per reaction were added into the individual reaction. LCN-HRM using the diluted 5% HCT116 and 5% NCI-H1650 was performed for KRAS exon 2 and EGFR exon 19 respectively. LCN-HRM positive reactions, which were detected by melting curve analysis, were directly sequenced. The first derivative melting plots and sequencing traces of two of the representative LCN-HRM positive reactions are shown. (Panel A for 5% HCT116 and Panel B for 5% NCI-H1650). The identical KRAS mutation to the HCT116, c.38G>A, was detected by sequencing of reactions 62 and 72, which displayed aberrant melting pattern compared with wild type. The EGFR delE746_A750 was detected heterozygously in reaction 66 and a homozygously in reaction 30 by sequencing.