Figure 1

Adaptation of Illumina TruSeq Custom Amplicon Kit to allow for single molecule tagging. (A) Schematic of method showing amplification of target DNA using custom probes and flanking primers. The P5-SMT primer is the same as the standard P5 index primer, but contains a degenerate 12 N-mer sequence in place of the index. The incorporation of an Ampure Bead size selection step after two rounds of PCR removes unused P5-SMT, and the P5 primer is added to facilitate downstream amplification. Figure schematic is adapted from Illumina promotional material. (B) Stacked barplot showing number of paired-end reads, split into unique reads (dark grey) and SMT-identified duplicate reads (light-grey) in 24 samples (18 germline, 6 tumor). (C) For each of 18 germline samples, we show the number of SMTs by duplicate cluster size (the number of times that an SMT was observed at a given target within a sample). Higher overall duplicate rates within a sample were associated with larger duplicate clusters. Except for the sample with the highest duplicate rate, duplicate cluster sizes were generally less than 10. The length of the SMT (8- versus 12-mer) did not affect the distribution. SMT, single molecule tag.