The UDT-Seq 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 partial Illumina adapter sequences to generate PCR amplicons in droplets, followed by the breaking of the emulsion and a secondary universal PCR amplification with primers that incorporate the remainder of the Illumina adaptor sequences. These amplicons are then directly sequenced on the Illumina platform for 2 × 125-nucleotide reads. This process removes the time consuming and error prone steps of sample fragmentation and library preparation, thus providing a streamlined process for easy implementation in the laboratory. In addition, as the direct sequencing approach (Figure S1b in Additional file 1) results in each base pair of an amplicon always being in the same position in a sequencing read, we are able to accurately estimate the position-dependent sequence read error rate, which is known to be variable in sequencing by synthesis [6]. This facilitates the sensitive and specific detection of low prevalence mutations in the tumor samples.
