The cancer mutational hotspots were selected from the COSMIC database v44 [14]. We initially selected all non-synonymous mutations involving single base substitutions and indels shorter than 140 bp in length. These criteria identified 9,935 mutations affecting 2,468 genes. The COSMIC database has some redundancy due to over-ascertainment of particular genes or cancer types; 96% of the mutations were observed less than 5 times and 2% were observed more than 100 times. Based on this clustering analysis, we chose 42 cancer genes (Table S1 in Additional file 2), which contain 53% (5271/9935) of all mutations and 87% (67,440/77,052) of all COSMIC (v44) valid entries (substitutions or small indels with reported genomic location). Only 141 of the 5,271 mutations were singletons defined as located more than 140 bp from another mutation (maximum length that can be assayed in one amplicon). We designed 518 primer pairs to amplify a total of 71 kb encompassing the 5,271 mutations.
