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 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 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 Cas9-Assisted Targeting of CHromosome segments (CATCH) for targeted nanopore sequencing and optical genome mapping

2017 ◽  
Author(s):  
Tslil Gabrieli ◽  
Hila Sharim ◽  
Yael Michaeli ◽  
Yuval Ebenstein
Keyword(s):  
Single Molecule ◽  
Genome Mapping ◽  
Single Point ◽  
Read Length ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Whole Genome Analysis ◽  
Long Read

ABSTRACTVariations in the genetic code, from single point mutations to large structural or copy number alterations, influence susceptibility, onset, and progression of genetic diseases and tumor transformation. Next-generation sequencing analysis is unable to reliably capture aberrations larger than the typical sequencing read length of several hundred bases. Long-read, single-molecule sequencing methods such as SMRT and nanopore sequencing can address larger variations, but require costly whole genome analysis. Here we describe a method for isolation and enrichment of a large genomic region of interest for targeted analysis based on Cas9 excision of two sites flanking the target region and isolation of the excised DNA segment by pulsed field gel electrophoresis. The isolated target remains intact and is ideally suited for optical genome mapping and long-read sequencing at high coverage. In addition, analysis is performed directly on native genomic DNA that retains genetic and epigenetic composition without amplification bias. This method enables detection of mutations and structural variants as well as detailed analysis by generation of hybrid scaffolds composed of optical maps and sequencing data at a fraction of the cost of whole genome sequencing.

scholarly journals Nanopore sequencing enables comprehensive transposable element epigenomic profiling

2020 ◽  
Author(s):  
Adam D. Ewing ◽  
Nathan Smits ◽  
Francisco J. Sanchez-Luque ◽  
Jamila Faivre ◽  
Paul M. Brennan ◽  
...  
Keyword(s):  
Transposable Element ◽  
Epigenetic Modifications ◽  
Cpg Methylation ◽  
Insertion Sequences ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Long Read ◽  
Paired Tumour

AbstractWe apply long-read nanopore sequencing and a new tool, TLDR (Transposons from Long Dirty Reads), to directly infer CpG methylation of new and extant human transposable element (TE) insertions in hippocampus, heart, and liver, as well as paired tumour and non-tumour liver. Whole genome TLDR analysis greatly facilitates studies of TE biology as complete insertion sequences and their epigenetic modifications are readily obtainable.

scholarly journals DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Liu ◽  
Wojciech Rosikiewicz ◽  
Ziwei Pan ◽  
Nathaniel Jillette ◽  
Ping Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Molecule ◽  
Evaluation Criteria ◽  
Systematic Evaluation ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Long Read ◽  
Genome Scale ◽  
Analytical Tools

Abstract Background Nanopore long-read sequencing technology greatly expands the capacity of long-range, single-molecule DNA-modification detection. A growing number of analytical tools have been developed to detect DNA methylation from nanopore sequencing reads. Here, we assess the performance of different methylation-calling tools to provide a systematic evaluation to guide researchers performing human epigenome-wide studies. Results We compare seven analytic tools for detecting DNA methylation from nanopore long-read sequencing data generated from human natural DNA at a whole-genome scale. We evaluate the per-read and per-site performance of CpG methylation prediction across different genomic contexts, CpG site coverage, and computational resources consumed by each tool. The seven tools exhibit different performances across the evaluation criteria. We show that the methylation prediction at regions with discordant DNA methylation patterns, intergenic regions, low CG density regions, and repetitive regions show room for improvement across all tools. Furthermore, we demonstrate that 5hmC levels at least partly contribute to the discrepancy between bisulfite and nanopore sequencing. Lastly, we provide an online DNA methylation database (https://nanome.jax.org) to display the DNA methylation levels detected by nanopore sequencing and bisulfite sequencing data across different genomic contexts. Conclusions Our study is the first systematic benchmark of computational methods for detection of mammalian whole-genome DNA modifications in nanopore sequencing. We provide a broad foundation for cross-platform standardization and an evaluation of analytical tools designed for genome-scale modified base detection using nanopore sequencing.

scholarly journals Transcriptional profile of platelets and iPSC-derived megakaryocytes from whole genome and RNA sequencing

Blood ◽  
2020 ◽  
Author(s):  
Kai Kammers ◽  
Margaret Taub ◽  
Benjamin Rodriguez ◽  
Lisa R Yanek ◽  
Ingo Ruczinski ◽  
...  
Keyword(s):  
Sequence Data ◽  
Blood Platelets ◽  
Induced Pluripotent Stem Cell ◽  
Normal Subjects ◽  
Blood Platelet ◽  
Tissue Expression ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Induced Pluripotent ◽  
Genomic Regions

GWAS studies have identified common variants associated with platelet related phenotypes, but because these variants are largely intronic or intergenic, their link to platelet biology is unclear. In 290 normal subjects from the GeneSTAR Research Study (110 African Americans (AAs) and 180 European Americans (EAs)), we generated whole genome sequence data from whole blood and RNA sequence (RNA-seq) data from extracted non-ribosomal RNA from 185 induced pluripotent stem cell-derived megakaryocyte (MK) cell lines (platelet precursor cells) and 290 blood platelet samples from these subjects. Using eigenMT software to select the peak SNP for each expressed gene, and meta-analyzing the results of AAs and EAs, we identify (q-value < 0.05) N=946 cis-expression quantitative trait loci (eQTLs) in derived MKs and N=1,830 cis-eQTLs in blood platelets. Among the 57 eQTLs shared between the two tissues the estimated directions of effect are very consistent (98.2% concordance). A high proportion of detected cis-eQTLs (74.9% in MKs and 84.3% in platelets) are unique to MKs and platelets compared to peak associated SNP-expressed gene pairs of 48 other tissue types that are reported in version V7 of the Genotype-Tissue Expression (GTEx) Project. The locations of our identified eQTLs are significantly enriched for overlap with several annotation tracks highlighting genomic regions with specific functionality in MKs, including MK-specific DNAse hotspots, H3K27-acetylation marks, H3K4-methylation marks, enhancers and super-enhancers. These results offer insights into the regulatory signature of MKs and platelets, with significant overlap in genes expressed, eQTLs detected, and enrichment within known super enhancers relevant to platelet biology.

scholarly journals Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

2020 ◽  
Vol 11 ◽  
Author(s):  
Frederico M. Batista ◽  
Tina Stapleton ◽  
James A. Lowther ◽  
Vera G. Fonseca ◽  
Rebecca Shaw ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Single Molecule ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Whole Genome ◽  
Nanopore Sequencing

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.

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