scholarly journals Oxford Nanopore sequencing-based protocol to detect CpG methylation in human mitochondrial DNA

2021 ◽  
Author(s):  
Iacopo Bicci ◽  
Claudia Calabrese ◽  
Zoe J. Golder ◽  
Aurora Gomez-Duran ◽  
Patrick F Chinnery
Keyword(s):  
Mitochondrial Dna ◽  
Single Molecule ◽  
Bisulfite Sequencing ◽  
Nuclear Dna ◽  
Cpg Methylation ◽  
Nanopore Sequencing ◽  
Single Molecule Level ◽  
Oxford Nanopore ◽  
Genome Bisulfite Sequencing ◽  
Technical Issues

SummaryMethylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered major technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns with WGBS, and then develop and assess the accuracy of a protocol for variant-specific methylation identification using long-read Oxford Nanopore Sequencing. Our approach circumvents mtDNA-specific confounders, while enriching for native full-length molecules over nuclear DNA. Variant calling analysis against Illumina deep re-sequencing showed that all expected mtDNA variants can be reliably identified. Methylation calling revealed negligible mtDNA methylation levels in multiple human primary and cancer cell lines. In conclusion, our protocol enables the reliable analysis of epigenetic modifications of mtDNA at single-molecule level at single base resolution, with potential applications beyond methylation.MotivationAlthough whole genome bisulfite sequencing (WGBS) is the gold-standard approach to determine base-level CpG methylation in the nuclear genome, emerging technical issues raise questions about its reliability for evaluating mitochondrial DNA (mtDNA) methylation. Concerns include mtDNA strand asymmetry rendering the C-rich light strand disproportionately vulnerable the chemical modifications introduced with WGBS. Also, short-read sequencing can result in a co-amplification of nuclear sequences originating from ancestral mtDNA with a high nucleotide similarity. Lastly, calling mtDNA alleles with varying proportions (heteroplasmy) is complicated by the C-to-T conversion introduced by WGBS on unmethylated CpGs. Here, we propose an alternative protocol to quantify methyl-CpGs in mtDNA, at single-molecule level, using Oxford Nanopore Sequencing (ONS). By optimizing the standard ONS library preparation, we achieved selective enrichment of native mtDNA and accurate single nucleotide variant and CpG methylation calling, thus overcoming previous limitations.

scholarly journals Minimal detection and low biological fluctuation of mitochondrial CpG methylation at the single-molecule level

2020 ◽  
Author(s):  
Chloe Goldsmith ◽  
Jesús Rafael Rodríguez-Aguilera ◽  
Ines El-Rifai ◽  
Adrien Jarretier ◽  
Valérie Hervieu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Single Molecule ◽  
Nuclear Dna ◽  
Mitochondrial Genes ◽  
Cpg Methylation ◽  
Nanopore Sequencing ◽  
Biological Processes ◽  
Single Molecule Level ◽  
Long Read ◽  
Low Levels

AbstractCytosine DNA methylation in the CpG context (5mCpG) is associated with the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mCpG has a regulatory role in this context. The main aim of this work was to develop and validate a novel tool for determining methylation of mtDNA and to corroborate its existence across different biological contexts. Using long-read nanopore sequencing we found low levels of CpG methylation (with few exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG was overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Although we do show several significant changes in all these conditions, 5mCpG is unlikely to play a major role in defining the transcriptional status of mitochondrial genes.

scholarly journals The SEQC2 epigenomics quality control (EpiQC) study

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jonathan Foox ◽  
Jessica Nordlund ◽  
Claudia Lalancette ◽  
Ting Gong ◽  
Michelle Lacey ◽  
...  
Keyword(s):  
Quality Control ◽  
Reference Materials ◽  
Single Molecule ◽  
Bisulfite Sequencing ◽  
Basic Research ◽  
Control Group ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Bisulfite Sequencing ◽  
High Concordance

Abstract Background Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group. Results Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. Conclusions The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

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

2021 ◽  
Vol 13 ◽  
Author(s):  
Theresa Lüth ◽  
Kobi Wasner ◽  
Christine Klein ◽  
Susen Schaake ◽  
Ronnie Tse ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Single Molecule ◽  
Induced Pluripotent Stem Cell ◽  
Cpg Methylation ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Biallelic Mutation ◽  
Synthetic Dna ◽  
Control Subjects ◽  
Induced Pluripotent

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.

scholarly journals Low biological fluctuation of mitochondrial CpG and non-CpG methylation at the single-molecule level

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chloe Goldsmith ◽  
Jesús Rafael Rodríguez-Aguilera ◽  
Ines El-Rifai ◽  
Adrien Jarretier-Yuste ◽  
Valérie Hervieu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Single Molecule ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
Nanopore Sequencing ◽  
High Coverage ◽  
Single Molecule Level ◽  
Modified Dna ◽  
Long Read ◽  
Low Levels

AbstractMammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.

scholarly journals Systematic benchmarking of tools for CpG methylation detection from Nanopore sequencing

2020 ◽  
Author(s):  
Zaka Wing-Sze Yuen ◽  
Akanksha Srivastava ◽  
Runa Daniel ◽  
Dennis McNevin ◽  
Cameron Jack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bisulfite Sequencing ◽  
Cpg Methylation ◽  
Genome Integrity ◽  
High Dispersion ◽  
Nanopore Sequencing ◽  
Consensus Approach ◽  
False Negatives ◽  
Control Of Gene Expression ◽  
Improved Accuracy

AbstractDNA methylation plays a fundamental role in the control of gene expression and genome integrity. Although there are multiple tools that enable its detection from Nanopore sequencing, their accuracy remains largely unknown. Here, we present a systematic benchmarking of tools for the detection of CpG methylation from Nanopore sequencing using individual reads, control mixtures of methylated and unmethylated reads, and bisulfite sequencing. We found that tools have a tradeoff between false positives and false negatives, and present a high dispersion with respect to the expected methylation frequency values. We described various strategies to improve the accuracy of these tools, including a new consensus approach, METEORE (https://github.com/comprna/METEORE), based on the combination of the predictions from two or more tools that shows improved accuracy over individual tools. Snakemake pipelines are provided for reproducibility and to enable the systematic application of our analyses to other datasets.

scholarly journals Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

2021 ◽  
Author(s):  
Romain Guitton ◽  
Christian Dölle ◽  
Guido Alves ◽  
Ole-Bjørn Tysnes ◽  
Gonzalo S. Nido ◽  
...  
Keyword(s):  
Gene Expression ◽  
Parkinson’S Disease ◽  
Mitochondrial Dna ◽  
Prefrontal Cortex ◽  
Cytosine Methylation ◽  
Bisulfite Sequencing ◽  
Whole Genome ◽  
Whole Genome Bisulfite Sequencing ◽  
Genome Bisulfite Sequencing ◽  
Low Levels

ABSTRACTWhile DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterised the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinson’s disease (n=27), using a combination of whole genome bisulfite sequencing (WGBS) and bisulfite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723±7,711), and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49±0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinson’s disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.Graphical abstract of the analyses and main findingsFresh-frozen brain tissue was obtained from the prefrontal cortex (Brodmann area 9) of 53 individuals, comprising 27 patients with idiopathic PD and 26 healthy controls. Tissue from the same samples was used in three different downstream analyses. WGBS was conducted on all 53 samples and the data were analysed using three different alignment strategies. Alignment against an mtDNA reference only was clearly superior as it gave the highest and most even depth of coverage. WGBS analysis revealed that mtDNA harbours very low levels of cytosine methylation, with the exception of the CpG position m.545 within the HSP1 region (lower right inset). The m.545 methylation was confirmed by bisulfite- and sequencing-independent methods, employing methylation-specific MspJI DNA digestion, followed by quantification with qPCR or fluorescent PCR and capillary electrophoresis. Finally, mtDNA transcript levels were determined by RT-qPCR and correlated to m.545 methylation levels, showing a positive association.

scholarly journals Experimental Confirmation of Multiple Co-Existent DNA Secondary Structures using Low-Yield Bisulfite Sequencing

2021 ◽  
Author(s):  
Jiaming Li ◽  
Jin Bae ◽  
Boyan Yordanov ◽  
Michael X Wang ◽  
Javier Gonzalez ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Single Molecule ◽  
Dna Sequences ◽  
Bisulfite Sequencing ◽  
Secondary Structures ◽  
Single Molecule Level ◽  
Dna Oligonucleotides ◽  
Metastable Structures ◽  
Structure State ◽  
Dna Secondary Structures

The prediction of DNA secondary structures from DNA sequences using thermodynamic models is imperfect for many biological sequences, both due to insufficient experimental data for training and to the kinetics of folding that lead to metastable structures. Here, we developed low-yield bisulfite sequencing (LYB-seq) to query the secondary structure states of cytosine (C) nucleotides in thousands of different DNA oligonucleotides on a single-molecule level. We observed that the reaction kinetics between bisulfite and C nucleotides is highly dependent on the secondary structure state of the C nucleotides, with the most accessible C nucleotides (those in small hairpin loops) reacting 70-fold faster than those in stable duplexes. Next, we developed a statistical model to evaluate the likelihood of an NGS read being consistent with a particular proposed secondary structure. By analyzing thousands of NGS reads for each DNA species, we can infer the distribution of secondary structures adopted by each species in solution. We find that 84% of 1,057 human genome subsequences studied here adopt 2 or more stable secondary structures in solution.

scholarly journals Systematic benchmarking of tools for CpG methylation detection from nanopore sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zaka Wing-Sze Yuen ◽  
Akanksha Srivastava ◽  
Runa Daniel ◽  
Dennis McNevin ◽  
Cameron Jack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bisulfite Sequencing ◽  
Cpg Methylation ◽  
Genome Integrity ◽  
High Dispersion ◽  
Nanopore Sequencing ◽  
Consensus Approach ◽  
False Negatives ◽  
Control Of Gene Expression ◽  
Improved Accuracy

AbstractDNA methylation plays a fundamental role in the control of gene expression and genome integrity. Although there are multiple tools that enable its detection from Nanopore sequencing, their accuracy remains largely unknown. Here, we present a systematic benchmarking of tools for the detection of CpG methylation from Nanopore sequencing using individual reads, control mixtures of methylated and unmethylated reads, and bisulfite sequencing. We found that tools have a tradeoff between false positives and false negatives and present a high dispersion with respect to the expected methylation frequency values. We described various strategies to improve the accuracy of these tools, including a consensus approach, METEORE (https://github.com/comprna/METEORE), based on the combination of the predictions from two or more tools that shows improved accuracy over individual tools. Snakemake pipelines are also provided for reproducibility and to enable the systematic application of our analyses to other datasets.

scholarly journals Analysis of mitochondrial genome methylation using Nanopore single-molecule sequencing

2021 ◽  
Author(s):  
Theresa Lüth ◽  
Christine Klein ◽  
Susen Schaake ◽  
Ronnie Tse ◽  
Sandro Pereira ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Single Molecule ◽  
Biological Significance ◽  
Cpg Methylation ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Low Level ◽  
Bisulfite Treatment ◽  
Synthetic Dna ◽  
Long Read

AbstractThe level and the biological significance of mitochondrial DNA (mtDNA) methylation in human cells is a controversial topic. Using long-read third-generation sequencing technology, mtDNA methylation can be detected directly from the sequencing data, which overcomes previously suggested biases, introduced by bisulfite treatment-dependent methods. We investigated mtDNA from whole blood-derived DNA and established a workflow to detect CpG methylation with Nanopolish. In order to obtain native mtDNA, we adjusted a whole-genome sequencing protocol and performed ligation library preparation and Nanopore sequencing. To validate the workflow, 897bp of methylated and unmethylated synthetic DNA samples at different dilution ratios were sequenced and CpG methylation was detected. Interestingly, we observed that reads with higher methylation in the synthetic DNA did not pass Guppy calling, possibly affecting conclusions about DNA methylation in Nanopore sequencing. We detected in all blood-derived samples overall low-level methylation across the mitochondrial genome, with exceptions at certain CpG sites. Our results suggest that Nanopore sequencing is capable of detecting low-level mtDNA methylation. However, further refinement of the bioinformatical pipelines including Guppy failed reads are recommended.

scholarly journals Multiplexed direct detection of barcoded protein reporters on a nanopore array

2019 ◽  
Author(s):  
Nicolas Cardozo ◽  
Karen Zhang ◽  
Katie Doroschak ◽  
Aerilynn Nguyen ◽  
Zoheb Siddiqui ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Direct Detection ◽  
Biological Activities ◽  
Sensor Technology ◽  
One Pot ◽  
Single Molecule Level ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
Reporter Proteins

AbstractGenetically encoded reporter proteins are a cornerstone of molecular biology. While they are widely used to measure many biological activities, the current number of uniquely addressable reporters that can be used together for one-pot multiplexed tracking is small due to overlapping detection channels such as fluorescence. To address this, we built an expanded library of orthogonally-barcoded Nanopore-addressable protein Tags Engineered as Reporters (NanoporeTERs), which can be read and demuxed by nanopore sensors at the single-molecule level. By adapting a commercially available nanopore sensor array platform typically used for real-time DNA and RNA sequencing (Oxford Nanopore Technologies’ MinION), we show direct detection of NanoporeTER expression levels from unprocessed bacterial culture with no specialized sample preparation. These results lay the foundations for a new class of reporter proteins to enable multiplexed, real-time tracking of gene expression with nascent nanopore sensor technology.

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