Custom‐Primed Rolling Circle Amplicons for Highly Accurate Nanopore Sequencing

Abstract Tandem repeats of a certain DNA sequence can be generated using rolling circle amplification (RCA), where a circular template is continuously amplified by a polymerase with strand displacement activity. In leveraging the linear repetition of the target sequence, enhanced accuracy is achievable by consensus calling in nanopore sequencing. However, traditional multiply‐primed RCA produces branched products with limited length, which may not be optimal for nanopore sequencing. In this study, an enhanced RCA protocol is introduced using sequence‐specific primers to produce longer and less branched amplicons. Taking advantage of the RCA amplicons of tandem repeats, custom‐primed rolling circle amplification sequencing (CPRSeq) is developed, a highly accurate nanopore sequencing pipeline. Utilizing CPRSeq, this successfully sequence standard samples of tumor‐associated single nucleotide variants at low mutation frequency and accomplished the whole‐genome sequencing and assembly of E. coli .