Reference : Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Inves...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
Systems Biomedicine
http://hdl.handle.net/10993/48897
Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept
English
Lüth, Theresa [Institute of Neurogenetics BMF, University of Lübeck and University Hospital Schleswig-Holstein Campus Lübeck, Lübeck, Germany]
Wasner, Kobi mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Molecular and Functional Neurobiology >]
Klein, Christine [Institute of Neurogenetics BMF, University of Lübeck and University Hospital Schleswig-Holstein Campus Lübeck, Lübeck, Germany]
Schaake, Susen [Institute of Neurogenetics BMF, University of Lübeck and University Hospital Schleswig-Holstein Campus Lübeck, Lübeck, Germany]
Tse, Ronnie [Institute of Neurogenetics BMF, University of Lübeck and University Hospital Schleswig-Holstein Campus Lübeck, Lübeck, Germany]
Pereira, Sandro L. [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)]
Laß, Joshua [Institute of Neurogenetics BMF, University of Lübeck and University Hospital Schleswig-Holstein Campus Lübeck, Lübeck, Germany]
Sinkkonen, Lasse mailto [University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM) >]
Grünewald, Anne mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Molecular and Functional Neurobiology >]
Trinh, Joanne [Institute of Neurogenetics BMF, University of Lübeck and University Hospital Schleswig-Holstein Campus Lübeck, Lübeck, Germany]
28-Sep-2021
Frontiers in Aging Neuroscience
Frontiers Media S.A.
Yes (verified by ORBilu)
International
1663-4365
1663-4365
Lausanne
Switzerland
[en] mitochondrial DNA ; methylation ; Nanopore
[en] 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.
German Research Foundation, Luxembourg National Research Fund, Personalized Medicine Consortium (PMC) of Luxembourg, European Community, Canadian Institutes of Health Research, Peter and Traudl Engelhorn Foundation, and the Joachim Herz Stiftung.
Researchers
http://hdl.handle.net/10993/48897
10.3389/fnagi.2021.713084
FnR ; FNR9631103 > Anne Grünewald > Model IPD > Modelling Idiopathic Parkinson’S Disease-associated Somatic Variation In Dopaminergic Neurons > 01/01/2016 > 31/12/2022 > 2015

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