Methplotlib: analysis of modified nucleotides from nanopore sequencing

Coster, Wouter De; Stražišar, Mojca

doi:10.1101/826107

Cited by 14 publications

(18 citation statements)

References 29 publications

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“…Previous works predicting DNA modifications from individual nanopore reads have typically relied on features such as signal intensity or dwell time to distinguish modified and unmodified read populations [46][47][48][49] . Here, in addition to these two features, we explored whether the use of 'trace' (also termed 'base probability'), which is reported directly by Guppy into the base-called FAST5 files, would improve our ability to predict RNA modification stoichiometry.…”

Section: Stoichiometry Prediction Of Y and Nm-modified Sites Using Simentioning

confidence: 99%

Quantitative profiling of native RNA modifications and their dynamics using nanopore sequencing

Begik

Lucas

Pryszcz

et al. 2020

Preprint

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SUMMARYA broad diversity of modifications decorate RNA molecules. Originally conceived as static components, evidence is accumulating that some RNA modifications may be dynamic, contributing to cellular responses to external signals and environmental circumstances. A major difficulty in studying these modifications, however, is the need of tailored protocols to map each modification individually. Here, we present a new approach that uses direct RNA nanopore sequencing to identify diverse RNA modification types present in native RNA molecules, using rRNA as the exemplar, and show that each RNA modification type results in distinct and characteristic base-calling ‘error’ signatures. We demonstrate the value of these signatures for de novo prediction of pseudouridine (Y) modifications transcriptome-wide, confirming known Y modifications in rRNAs, snRNAs and mRNAs, and uncovering a novel Pus4-dependent Y modification in yeast mitochondrial rRNA. Using a machine learning classifier, we show that the stoichiometry of modified sites can be quantified by identifying current intensity alterations in individual RNA reads. Finally, we explore the dynamics of pseudouridylation across a battery of environmental stresses, revealing novel heat-sensitive Y-modified sites in both snRNAs and snoRNAs. Altogether, our work demonstrates that Y RNA modifications can be predicted de novo and in a quantitative manner using native RNA nanopore sequencing.

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Section: Stoichiometry Prediction Of Y and Nm-modified Sites Using Simentioning

confidence: 99%

Quantitative profiling of native RNA modifications and their dynamics using nanopore sequencing

Begik

Lucas

Pryszcz

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Indicated are the modification likelihoods (red, high; blue, low), the modification frequency, which ranges from 0 to 1, and the annotation that is based on the (complemented) reference genomes (HG38 and Mmul_10). Plots were generated by Methplotlib (De Coster et al 2020). The KIR promotor region, which is approximately 300 bp in front of exon 1 (Chan et al 2003), is highly modified for human KIR3DS1 (A) and macaque KIR3DLW03 (B), with modification frequencies ranging from 85 to 95%.…”

Section: Resultsmentioning

confidence: 99%

“…The basecalled reads were mapped to a reference genome using minimap2, and subsequently called for modifications using Nanopolish. The methylation likelihood and frequencies were visualized by Methplotlib (De Coster et al 2020), using an annotated reference genome. When the reference genome did not contain the appropriate KIR genes, like Mmul_10 for all macaque individuals, methylation calls were annotated using a modified reference genome, which was complemented with the newly assembled haplotypes as artificial chromosomes.…”

Section: Methodsmentioning

confidence: 99%

Rapid characterization of complex genomic regions using Cas9 enrichment and Nanopore sequencing

Bruijnesteijn

Wiel

Groot

et al. 2021

Preprint

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Long-read sequencing approaches have considerably improved the quality and contiguity of genome assemblies. Such platforms bear the potential to resolve even extremely complex regions, such as multigenic families and repetitive stretches of DNA. Deep sequencing coverage, however, is required to overcome low nucleotide accuracy, especially in regions with high homopolymer density, copy number variation, and sequence similarity, such as the MHC and KIR gene clusters of the immune system. Therefore, we have adapted a targeted enrichment protocol in combination with long-read sequencing to efficiently annotate complex genomic regions. Using Cas9 endonuclease activity, segments of the complex KIR gene cluster were enriched and sequenced on an Oxford Nanopore Technologies platform. This provided sufficient coverage to accurately resolve and phase highly complex KIR haplotypes. Our strategy facilitates rapid characterization of large and complex multigenic regions, including its epigenetic footprint, in multiple species, even in the absence of a reference genome.

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“…The Benjamini-Hochberg procedure was applied to t-test p-values for false discovery rate (FDR) correction at ⍺ = 0.05. The Python tool methplotlib v. 0.1.1 (De Coster et al 2020) was used to visualize regions of interest graphically and to further summarize data.…”

Section: Methodsmentioning

confidence: 99%

Dna Methylation Profiling of a Cnidarian-Algal Symbiosis Using Nanopore Sequencing

Dimond

Roberts

Nguyen

2021

Preprint

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Symbiosis with protists is common among cnidarians such as corals and sea anemones, and is associated with homeostatic and phenotypic changes in the host that could have epigenetic underpinnings, such as methylation of CpG dinucleotides. We leveraged the sensitivity to base modifications of nanopore sequencing to probe the effect of symbiosis with the chlorophyte Elliptochloris marina on methylation in the sea anemone Anthopleura elegantissima. We first validated the approach by comparison of nanopore-derived methylation levels with CpG depletion analysis of a published transcriptome, finding that high methylation levels are associated with CpG depletion as expected. Next, using reads generated exclusively from aposymbiotic anemones, a largely complete draft genome comprising 243 Mb was assembled. Reads from aposymbiotic and symbiotic sea anemones were then mapped to this genome and assessed for methylation using the program Nanopolish, which detects signal disruptions from base modifications as they pass through the nanopore. Based on assessment of 452,841 CpGs for which there was adequate read coverage (approximately 8% of the CpGs in the genome), symbiosis with E. marina was, surprisingly, associated with only subtle changes in the host methylome. However, we did identify one extended genomic region with consistently higher methylation among symbiotic individuals. The region was associated with a DNA polymerase zeta that is noted for its role in translesion synthesis, which opens interesting questions about the biology of this symbiosis. Our study highlights the power and relative simplicity of nanopore sequencing for studies of nucleic acid base modifications in non-model species.

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Methplotlib: analysis of modified nucleotides from nanopore sequencing

Cited by 14 publications

References 29 publications

Quantitative profiling of native RNA modifications and their dynamics using nanopore sequencing

Quantitative profiling of native RNA modifications and their dynamics using nanopore sequencing

Rapid characterization of complex genomic regions using Cas9 enrichment and Nanopore sequencing

Dna Methylation Profiling of a Cnidarian-Algal Symbiosis Using Nanopore Sequencing

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