The UDT-Seq assay is a direct sequencing method of approximately 200-nucleotide long PCR amplicons generated in multiplex using microdroplet PCR [13] (Figure S1a in Additional file 1). Briefly, we use chimeric primer pairs, containing both locus-specific and adapter sequences, to generate PCR amplicons that are then directly sequenced on the Illumina Genome Analyzer II (GAII) platform for 2 × 125-nucleotide reads. This process simplifies the workflow by removing the time consuming and error prone step of sample fragmentation and library preparation, thus providing a streamlined process for easy implementation in the laboratory. In addition, as the direct sequencing approach results in each base pair of an amplicon always being in the same position in a sequencing read (Figure S1b in Additional file 1), we are able to accurately measure the position-dependent sequencing error rate, which is known to vary significantly 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
