Somatic variants can be confirmed in unamplified cell source material by (i) targeted DNA capture followed by high-coverage (>100x) DNA resequencing, (ii) high-coverage sequencing of multiplexed PCR amplicons, and (iii) droplet digital PCR (ddPCR). These approaches vary in throughput and sensitivity. Targeted DNA capture and resequencing can require the creation of several thousand custom oligonucleotides designed to capture the genomic DNA either including or surrounding the putative variants. The captured DNA then is subjected to high-coverage paired-end DNA sequencing, yielding a typical sensitivity of variant detection in greater than 1% of cells. Amplicon sequencing involves PCR amplification of candidate loci followed by high-coverage paired-end DNA sequencing, yielding a typical sensitivity of variant detection in greater than 0.1% of cells. Finally, ddPCR involves partitioning a DNA sample into large numbers of individual droplets that generally contain one copy of template DNA. PCR takes place within these droplets, leading to the production of a fluorescent readout, either through the use of an intercalating dye or a fluorescent oligomer probe, to indicate the presence or absence of the PCR target of interest. Subsequent
