We have applied DS to a number of biological systems, including the M13mp2 phagemid19, human mtDNA from frozen brain tissue33 and human nuclear DNA from both frozen and formalin-fixed samples (M.J.P., E.J.F. and L.A.L.; unpublished results). We consistently observe a >50,000-fold reduction in the apparent mutation frequency in these samples, relative to conventional NGS methods. We have found that SSCS formation is capable of removing almost all sequencer-derived false positives, which account for >99% of all artifacts. However, this step is unable to remove artifacts arising from first-round PCR errors that have been propagated to all tag family members. The mutational spectrum of the SSCS typically shows a high frequency of G®T mutations, which is the result of fixation of oxidative damage occurring during the DNA purification and processing steps19. Formation of the DCS reads consistently removes these artifacts, as well as those from other damage events, and it results in a further 90–99+% reduction in mutational artifacts.
