As the need for accurate sequencing methods has increased, three main strategies besides DS have emerged: (i) single-cell sequencing4,27-29, (ii) single-stranded molecular barcoding20,21,23 and (iii) circle sequencing (CirSeq)30,31. Although each approach has unique strengths, all three of these methods involve sequencing DNA derived from a single strand of a double-stranded molecule. Misincorporation events by DNA polymerase occurring during the first round of amplification will inherently be propagated to the daughter molecules, and they are likely to be erroneously scored as mutations. In the case of single-cell sequencing, the use of random primer sequences in conjunction with a strand-displacing DNA polymerase results in random priming from the newly synthesized DNA and the generation of ‘copies of copies’, thus propagating any initial misincorporation events to all of the reads. Because all the reads are derived from a single cell, the propagated error would be incorrectly called a genetic variant. A similar process can occur in CirSeq and singlestranded barcoding, whereby a misinsertion event occurring during the first round of synthesis can be propagated to subsequently synthesized daughter molecules. When sequenced, these subpopulations
