Analysis of mitochondrial genome methylation using Nanopore single-molecule sequencing

Lueth, Theresa; Klein, Christine; Schaake, Susen; R, Tse; Pereira, Sofía; Laß, Joshua; Sinkkonen, Lasse; Gruenewald, Anne; Trinh, Joanne

doi:10.1101/2021.02.05.429923

Cited by 5 publications

(4 citation statements)

References 31 publications

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“…Here we report essentially zero DNA methylation at every CpG position in the mitogenome in stark contrast to other reports of low but consistent methylation in mtDNA. Mitochondrial methylation using a nanopore sequencer has been reported by at least four groups [6,[29][30][31]. All of these studies found low but measurable mtDNA methylation, averaging less than 7% across a variety of conditions and cell types, though a few specific CpG sites had over 20%.…”

Section: Discussionmentioning

confidence: 96%

The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing

et al. 2021

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Background The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. Results Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3′ side of several coding regions. Conclusions Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.

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Section: Discussionmentioning

confidence: 96%

The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing

et al. 2021

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“…Here we report essentially zero DNA methylation at every CpG position in the mitogenome in stark contrast to other reports of low but consistent methylation in mtDNA. Mitochondrial methylation using a nanopore sequencer has been reported by at least four groups (Aminuddin et al 2020;Bicci et al 2021;Lüth et al 2021;Goldsmith et al 2020). All of these studies found low but measurable mtDNA methylation, averaging less than 7% across a variety of conditions and cell types, though a few specific CpG sites had over 20%.…”

Section: Discussionmentioning

confidence: 96%

The Mitochondrial Genome and Epigenome of the Golden Lion Tamarin from Fecal DNA using Nanopore Adaptive Sequencing

Wanner

Larsen

McLain

et al. 2021

Preprint

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The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3' side of several coding regions. Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.

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“…We acknowledge Aurelien Ginolhac for his review of the manuscript and insightful comments and Philip Seibler for expertise in IPSC-derived neuronal models. The study is available as a preprint (Lüth et al, 2021 ).…”

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confidence: 99%

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept

Lüth

Wasner

Klein

et al. 2021

Front. Aging Neurosci.

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Objective: To establish a workflow for mitochondrial DNA (mtDNA) CpG methylation using Nanopore whole-genome sequencing and perform first pilot experiments on affected Parkin biallelic mutation carriers (Parkin-PD) and healthy controls.Background: Mitochondria, including mtDNA, are established key players in Parkinson's disease (PD) pathogenesis. Mutations in Parkin, essential for degradation of damaged mitochondria, cause early-onset PD. However, mtDNA methylation and its implication in PD is understudied. Herein, we establish a workflow using Nanopore sequencing to directly detect mtDNA CpG methylation and compare mtDNA methylation between Parkin-related PD and healthy individuals.Methods: To obtain mtDNA, whole-genome Nanopore sequencing was performed on blood-derived from five Parkin-PD and three control subjects. In addition, induced pluripotent stem cell (iPSC)-derived midbrain neurons from four of these patients with PD and the three control subjects were investigated. The workflow was validated, using methylated and unmethylated 897 bp synthetic DNA samples at different dilution ratios (0, 50, 100% methylation) and mtDNA without methylation. MtDNA CpG methylation frequency (MF) was detected using Nanopolish and Megalodon.Results: Across all blood-derived samples, we obtained a mean coverage of 250.3X (SD ± 80.5X) and across all neuron-derived samples 830X (SD ± 465X) of the mitochondrial genome. We detected overall low-level CpG methylation from the blood-derived DNA (mean MF ± SD = 0.029 ± 0.041) and neuron-derived DNA (mean MF ± SD = 0.019 ± 0.035). Validation of the workflow, using synthetic DNA samples showed that highly methylated DNA molecules were prone to lower Guppy Phred quality scores and thereby more likely to fail Guppy base-calling. CpG methylation in blood- and neuron-derived DNA was significantly lower in Parkin-PD compared to controls (Mann-Whitney U-test p < 0.05).Conclusion: Nanopore sequencing is a useful method to investigate mtDNA methylation architecture, including Guppy-failed reads is of importance when investigating highly methylated sites. We present a mtDNA methylation workflow and suggest methylation variability across different tissues and between Parkin-PD patients and controls as an initial model to investigate.

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Analysis of mitochondrial genome methylation using Nanopore single-molecule sequencing

Cited by 5 publications

References 31 publications

The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing

The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing

The Mitochondrial Genome and Epigenome of the Golden Lion Tamarin from Fecal DNA using Nanopore Adaptive Sequencing

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept

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