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Abstract BackgroundDirect-sequencing technologies, such as Oxford Nanopore’s, are delivering long RNA reads with great efficacy and convenience. These technologies afford an ability to detect post-transcriptional modifications at a single-molecule resolution, promising new insights into the functional roles of RNA. However, realizing this potential requires new tools to analyze and explore this type of data. ResultHere, we present Sequoia, a visual analytics tool that allows users to interactively explore nanopore sequences. Sequoia combines a Python-based backend with a multi-view visualization interface, enabling users to import raw nanopore sequencing data in a Fast5 format, cluster sequences based on electric-current similarities, and drill-down onto signals to identify properties of interest. We demonstrate the application of Sequoia by generating and analyzing ~ 500k reads from direct RNA sequencing data of human HeLa cell line. We focus on comparing signal features from m6A and m5C RNA modifications as the first step towards building automated classifiers. We show how, through iterative visual exploration and tuning of dimensionality reduction parameters, we can separate modified RNA sequences from their unmodified counterparts. We also document new, qualitative signal signatures that characterize these modifications from otherwise normal RNA bases, which we were able to discover from the visualization. ConclusionsSequoia’s interactive features complement existing computational approaches in nanopore-based RNA workflows. The insights gleaned through visual analysis should help users in developing rationales, hypotheses, and insights into the dynamic nature of RNA. Sequoia is available athttps://github.com/dnonatar/Sequoia.
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Uncalled4 improves nanopore DNA and RNA modification detection via fast and accurate signal alignment
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.
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- Award ID(s):
- 2216612
- PAR ID:
- 10579555
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Methods
- Volume:
- 22
- Issue:
- 4
- ISSN:
- 1548-7091
- Format(s):
- Medium: X Size: p. 681-691
- Size(s):
- p. 681-691
- Sponsoring Org:
- National Science Foundation
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