CLAE: A High‐Fidelity Nanopore Sequencing Strategy for Read‐Level Viral Variant Detection and Environmental RNA Virus Discovery

High‐fidelity (HF) long‐read sequencing enables accurate profiling of microorganisms and pathogens at single‐molecule resolution. However, current Oxford Nanopore Technologies (ONT)—a revolutionary platform offering real‐time, portable sequencing at relatively low instrumental cost—suffer from severe read‐length bias, limited accuracy (often <Q20), and low throughput. Here, Circular‐ and Linear‐Amplicon‐Mediated Error Correction (CLAE) is introduced, a biochemical and computational approach that addresses these limitations by integrating hairpin ligation, pre‐circling, single‐stranded DNA linearization, and targeted nickase‐based debranching. CLAE significantly enhances rolling‐circle amplification (RCA) efficiency for long DNA templates, markedly improving Nanopore sequencing yield and accuracy. CLAE achieves Q30‐level accuracy in up to 27% of RCA reads, throughput exceeding 800 Mb per 100 pores, and an N50 of ≈15 Kb (bacterial genome). Moreover, its bidirectional subreads and high throughput substantially boost accuracy without compromising read length. CLAE is validated by resolving SARS‐CoV‐2 quasi‐species from community wastewater and recovering novel, full‐length RNA virus genomes from marine samples. CLAE enables precise variant detection in complex samples and corrects short‐read misassemblies, significantly broadening ONT's utility in metaviromics, epidemiology, and environmental surveillance. Thus, CLAE establishes a versatile, field‐compatible platform for high‐fidelity viral genome sequencing in targeted and agnostic contexts.