There is growing need for technologies capable of resolving subclonal mutations. For example, genetic heterogeneity has long been proposed to be an intrinsic driver of cancer initiation and progression2. Recent tumor genome sequencing studies suggest that human cancers exhibit extreme levels of genetic heterogeneity3-6. Subclonal mutations are probably a major factor in cancer relapse and in rapid emergence of chemotherapy resistance7-9. However, the study of cancer subclones requires the confident detection of mutations that are present in <1% of cells—a level of resolution that cannot be obtained by conventional sequencing approaches. Similarly, the genetic diversity found within microbial populations underlies their ability to adapt to changing environments, including development of drug resistance10-13, but this genetic diversity is difficult to directly assess owing to the high background error rate of conventional NGS sequencing. Other fields with a similar need for robust low-frequency mutation detection include forensics14, paleogenomics15,16, evolution17 and toxicology18, as high-accuracy sequencing would allow one to assess the potential mutagenicity of new chemical compounds without the need for a genetic selection system to identify mutant genes.
