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

Theresa Lüth,Susen Schaake,Anne Grünewald,Lasse Sinkkonen,Kobi Wasner,Ronnie Tse,Christine Klein,Sandro L Pereira,Joanne Trinh,Joshua Laß

doi:10.3389/fnagi.2021.713084

Abstract

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.

Highlights

Given the importance of environmental impact on mtDNA and its association with disease, the investigation of mtDNA CpG methylation is of interest
Our results suggest that mtDNA methylation exists and that it can be reliably detected by Nanopore sequencing
We obtained a mean of 8.6 Gb of data and a read length of 4.9 kb with whole-genome Nanopore sequencing

Summary

Introduction

Given the importance of environmental impact on mtDNA and its association with disease, the investigation of mtDNA CpG methylation is of interest. The secondary and tertiary structure of intact mtDNA may block cytosines from bisulfite conversion, which influences the outcome of the detected methylation levels (Mechta et al, 2017; Owa et al, 2018). Bisulfite conversion-resistant cytosines can lead to an overestimation of mtDNA methylation. For linearized mtDNA prior to bisulfite treatment, lower levels of methylation have been observed compared to intact mtDNA (Liu et al, 2016; Mechta et al, 2017). We establish a workflow using Nanopore sequencing to directly detect mtDNA CpG methylation and compare mtDNA methylation between Parkin-related PD and healthy individuals

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Results

Discussion

Conclusion