SWARM: A Single-Molecule Workflow for High-Precision Profiling of RNA Modifications
Nanopore direct RNA sequencing promises to decode the epitranscriptome by detecting multiple modifications on individual RNA molecules, but its potential for biological discovery is hampered by high false-positive rates. We present SWARM, an AI-based framework designed to overcome this fundamental limitation. Its key innovation is a training strategy incorporating non-target modifications to eliminate modification crosstalk, enabling high-precision detection of m6A, pseudouridine (?), and m5C at single-nucleotide and single-molecule resolution from a single sample. Using rigorous in-vitro and cellular RNA benchmarks, SWARM outperforms existing tools and shows strong agreement with orthogonal methods. Applying SWARM across mammalian tissues yielded thousands of novel modification sites with confirmed motifs and localisation patterns. Our high-resolution multi-tissue modification map revealed no evidence of widespread m6A-? interplay, challenging models of a coordinated epitranscriptomic code. We further discovered splicing-shaped pseudouridylation, where TRUB1-mediated modification occurs only after exon-exon ligation stabilises the substrate structure. SWARM provides a robust, universally applicable tool for epitranscriptome discovery.
Nanopore direct RNA sequencing promises to decode the epitranscriptome by detecting multiple modifications on individual RNA molecules, but its potential for biological discovery is hampered by high false-positive rates. We present SWARM, an AI-based framework designed to overcome this fundamental limitation. Its key innovation is a training strategy incorporating non-target modifications to eliminate modification crosstalk, enabling high-precision detection of m6A, pseudouridine (?), and m5C at single-nucleotide and single-molecule resolution from a single sample. Using rigorous in-vitro and cellular RNA benchmarks, SWARM outperforms existing tools and shows strong agreement with orthogonal methods. Applying SWARM across mammalian tissues yielded thousands of novel modification sites with confirmed motifs and localisation patterns. Our high-resolution multi-tissue modification map revealed no evidence of widespread m6A-? interplay, challenging models of a coordinated epitranscriptomic code. We further discovered splicing-shaped pseudouridylation, where TRUB1-mediated modification occurs only after exon-exon ligation stabilises the substrate structure. SWARM provides a robust, universally applicable tool for epitranscriptome discovery.
John Curtin School of Medical Research