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Abstract Nanopore signal analysis enables detection of nucleotide modifications from native DNA and RNA sequencing, providing both accurate genetic or transcriptomic and epigenetic information without additional library preparation. At present, only a limited set of modifications can be directly basecalled (for example, 5-methylcytosine), while most others require exploratory methods that often begin with alignment of nanopore signal to a nucleotide reference. We present Uncalled4, a toolkit for nanopore signal alignment, analysis and visualization. Uncalled4 features an efficient banded signal alignment algorithm, BAM signal alignment file format, statistics for comparing signal alignment methods and a reproducible de novo training method fork-mer-based pore models, revealing potential errors in Oxford Nanopore Technologies’ state-of-the-art DNA model. We apply Uncalled4 to RNA 6-methyladenine (m6A) detection in seven human cell lines, identifying 26% more modifications than Nanopolish using m6Anet, including in several genes where m6A has known implications in cancer. Uncalled4 is available open source atgithub.com/skovaka/uncalled4.