Custom-primed rolling circle amplicons for highly accurate nanopore
Rolling circle amplification(RCA) is a method for generating tandem repeats. We presented an enhanced RCA protocol to generate less branched amplicons with longer products by using sequence-specific primers. More tandem repeats of RCA amplicons were applied to nanopore sequencing for highly accurate sequencing, which was CPRSeq. CPRSeq can be applied for accurately sequencing various dsDNA samples, such short dsDNA fragments or genome. Here, we have reported two applications based on CPRSeq, mutation detection and E. coli genome sequencing. Fistly, in the application of mutation detection, we have detected a gradient mutation frequency of three standard samples (5%, 3%, 1%, 0.5%, 0.1%) respectively. The three mutation samples are EGFR L858R, KRAS 12C and KRAS 13D. Secondly, in the application of genome sequencing, we detected E. coli genome by nanopore sequencer and aseembly (template-free). With increasing repeats of amplicons from RCA increasing, sequencing accuracy was increased. And when the repeats were 10, the sequencing accuracy was chosen to guarantee an accuracy level.