To overcome the high error rate of next-generation sequencing and thereby facilitate the study of subclonal and random mutations, we recently developed a highly sensitive sequencing methodology termed Duplex Sequencing (DS). DS yields unprecedented accuracy in sequencing of double-stranded DNA, with a >10,000-fold improvement compared with conventional NGS, and it has the unique ability to detect a single mutation among >107 sequenced bases19. DS takes advantage of the inherent complementarity of double-stranded DNA by using degenerate molecular tags20-26 to label each fragmented DNA molecule with its own unique DNA sequence. By tagging duplex DNA with adapters containing random yet complementary double-stranded nucleotide sequences, it becomes feasible to trace every sequence read back to one of the two strands of the original double-stranded DNA molecule (Fig. 1a). After adapter ligation, the individually labeled strands are PCR-amplified to create sequence families that share the same tag sequences derived from each of the two single parental strands (Fig. 1b). After sequencing, members of each tag family are grouped and a consensus sequence is established for each of the two strands to form ‘single-strand
