Custom‐Primed Rolling Circle Amplicons for Highly Accurate Nanopore Sequencing

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.