DS is a uniquely sensitive method that is capable of detecting and quantifying mutations that occur at extremely low frequencies; however, this ability comes at a cost. Owing to the method’s reliance on sequencing multiple PCR duplicates of both DNA strands, DS requires much larger sequencing capacity than conventional NGS to produce a given depth of sequencing. At present, the use of current NGS technology with DS to sequence large genomes or genome targets >1–2 Mbp in size to a high depth of coverage is prohibitively expensive; however, as the cost of sequencing continues to fall, we anticipate that this will become increasingly practical.
