in sequencing by synthesis [6]. This facilitates the sensitive and specific detection of low prevalence mutations in the tumor samples. The cancer mutational hotspots screened by UDT-Seq were selected from the COSMIC database v44 [14]. An unsupervised clustering analysis (Materials and methods) led to the identification of cancer hotspots in 42 cancer genes (Table S1 in Additional file 2), which contain 53% (5,271/9,935) of all mutations and 87% (67,440/77,052) of all COSMIC database valid entries (substitutions or small indels with reported genomic location). We designed 518 primer pairs (Table S2 in Additional file 2) to amplify a total of 71.1 kb encompassing these cancer mutational hotspots.
