Zhang Jiayi, Yang Xujuan, Xuan Weijia, Wu Zhiguang, Ding Jiawei, Wang Zhenqi, Yan Qi, Zhang Tianqing, Qi Zhi, Bai Jingwei, Wei Bryan
School of Life Sciences, Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China.
School of Pharmaceutical Sciences, Tsinghua University, Beijing, 100084, China.
Small Methods. 2025 Jun;9(6):e2401416. doi: 10.1002/smtd.202401416. Epub 2025 Mar 3.
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.
特定DNA序列的串联重复可以通过滚环扩增（RCA）产生，其中圆形模板由具有链置换活性的聚合酶持续扩增。利用目标序列的线性重复，通过纳米孔测序中的一致性调用可以实现更高的准确性。然而，传统的多重引物RCA会产生长度有限的分支产物，这可能不适用于纳米孔测序。在本研究中，引入了一种增强的RCA方案，使用序列特异性引物来产生更长且分支更少的扩增子。利用串联重复的RCA扩增子，开发了定制引物滚环扩增测序（CPRSeq），这是一种高度准确的纳米孔测序流程。利用CPRSeq，成功地对低突变频率的肿瘤相关单核苷酸变异的标准样本进行了测序，并完成了大肠杆菌的全基因组测序和组装。
