Nanopore sequencing and assembly of a human genome with ultra-long reads. Given the importance of environmental impact on mtDNA and its association with disease, the investigation of mtDNA CpG methylation is of interest.
Thus, the software predicting the MF tools might need to be trained with more mtDNA-specific data sets to enhance the accuracy even more. To update your cookie settings, please visit the. The difference was present using either Nanopolish or Megalodon. Bisulfite conversion-resistant cytosines can lead to an overestimation of mtDNA methylation. 
also referred to as continuous long read (CLR) or polymerase read, all of which are terms for the single long read coming off a PacBio including adaptors and insert sequence. Strategies for the Enrichment and Selection of Genetically Modified Cells, Academic & Personal: 24 hour online access, Corporate R&D Professionals: 24 hour online access, https://doi.org/10.1016/j.tibtech.2018.07.013, Single-Molecule Sequencing: Towards Clinical Applications, https://doi.org/10.1016/j.tig.2018.05.008, For academic or personal research use, select 'Academic and Personal', For corporate R&D use, select 'Corporate R&D Professionals'. There are multiple copies of the 45S rRNA gene in the human genome, comparable in size and coverage to the mtDNA. 57 Articles, This article is part of the Research Topic, https://doi.org/10.3389/fnagi.2021.713084, https://github.com/nanoporetech/megalodon, https://www.frontiersin.org/articles/10.3389/fnagi.2021.713084/full#supplementary-material, Creative Commons Attribution License (CC BY). consensus sequence of one or more complete or partial passes of the insert on a PacBio platform. Comparison of mtDNA CpG methylation between blood- and neuron-derived DNA. In these studies, the coverage of the mitochondrial genome ranged from 60.4X (Aminuddin et al., 2020) to over 10000X (Goldsmith et al., 2021) (Supplementary Table 3). We analyzed the relationship between blood- and neuron-derived mtDNA of each CpG site for individual participants and observed that the methylation frequency in blood- and neuron-derived mtDNA was correlated (Spearman's r > 0.5659, Spearman's exploratory p-value < 0.0001, Supplementary Figure 9). All authors participated in revising the manuscript for intellectual content and approved its final version. By contrast, future studies should be based on fresh blood draws to allow for a correlation of cell type and mtDNA methylation status in a given sample. In addition, all sites that showed a methylation frequency >0.2 in a PCR product were excluded from downstream analyses. PLoS ONE 11:e0167600. We explored possible NUMTs contamination in our data. Parkinsons disease associated with pure ATXN10 repeat expansion. Nucleic Acids Res. Figure 2. doi: 10.1002/mds.27559, van der Wijst, M. G., Van Tilburg, A. Y., Ruiters, M. H., and Rots, M. G. (2017). Lastly, we analyzed the relationship between the age at examination of the participants and the overall methylation level on the mtDNA. 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. Conclusion: Nanopore sequencing is a useful method to investigate mtDNA methylation architecture, including Guppy-failed reads is of importance when investigating highly methylated sites. Hence, rare variants or variants not associated with their traditional haplotype can be assigned properly to each allele. As nuclear genome methylation is associated with age (Benayoun et al., 2015; Jiang and Guo, 2020) and similar findings have been reported for mtDNA (Mawlood et al., 2016), we analyzed the relationship between age and mtDNA CpG methylation in both, the blood and neuronal samples, but did not observe a correlation. 48:e88. JAFFA: high sensitivity transcriptome-focused fusion gene detection. The flow cell was primed and loaded according to the manufacturer's instructions. ACMG clinical laboratory standards for next-generation sequencing. MtDNA CpG methylation frequency (MF) was detected using Nanopolish and Megalodon. repetitive DNA contents of clinical utility. Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.
Background: Mitochondria, including mtDNA, are established key players in Parkinson's disease (PD) pathogenesis. Flexible and scalable full-length CYP2D6 long amplicon PacBio sequencing. Piercing the dark matter: bioinformatics of long-range sequencing and mapping. Messenger RNA modifications: form, distribution, and function. N2/B27 medium with 200 M AA, 1 M PMA and 100 ng FGF8 (PeproTech) was used for eight days and then for two days N2/B27 medium with 200 M AA and 0.5 M PMA. Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells. To investigate the possibility of preferential sequencing of unmethylated reads and methylated reads, which do not pass the quality threshold during base-calling, we analyzed Guppy-failed reads. To consider the results obtained from the investigation of the synthetic DNA samples, mtDNA methylation was re-analyzed, including Guppy-passed and -failed reads. In addition, there was a negative correlation between coverage of CpG sites and detected MF with Nanopolish from passed reads in the 100% methylated sample (Spearman's r = 0.2884, Spearman's exploratory p-value = 0.0381, Figure 4D) and in contrast, there was a trend toward a positive association when the MF was detected from the failed reads (Spearman's r = 0.2687, Spearman's exploratory p-value > 0.05, Figure 4E). Mitochondrial and 45S rRNA CpG methylation comparison by strand. In addition, a small decrease of ND1 methylation was observed which led to an increase in ND1 expression (Blanch et al., 2016). Radar chart showing mitochondrial DNA methylation from eight blood-derived DNA samples detected by Nanopolish. Overall, we detected low-level CpG methylation of mtDNA with exceptions for certain sites. Nanopore sequencing was performed with the MinION or GidION using the R9.4.1 flow cells. You will then receive an email that contains a secure link for resetting your password, If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password. 
Five of these individuals are PD patients with Parkin mutations (Parkin-PD), with three being compound-heterozygous carriers (L-3244, L-13062 and B-11) and two homozygous carriers (L-3048 and B-125) (Supplementary Table 1). The CpG MF detected from the mtDNA plus- and minus-strands was corrected for the FPR, as well. 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. R2C2: improving nanopore read accuracy enables the sequencing of highly-multiplexed full-length single-cell cDNA. Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT sequencing of repeat-expansion disease causative genomic regions. J. Mol. The blood-derived mtDNA mean MF detected with Nanopolish in Parkin-PD was 0.027 (SD = 0.038) compared to 0.034 (SD = 0.047) in healthy controls (Mann Whitney U-test p = 0.0002, Figure 6A) and with Megalodon the mean MF was 0.036 (SD = 0.051) in patients compared to 0.048 (SD = 0.065) in control subjects (Mann Whitney U-test p < 0.0001, Figure 6B). No use, distribution or reproduction is permitted which does not comply with these terms. Nanopolish reports the log-likelihood ratio for each observed event (i.e., CpG site within a k-mer sequence). (2016). sequencing nextbigfuture Distribution and dynamics of mitochondrial DNA methylation in oocytes, embryos and granulosa cells. sequencing nanopore advances rna
When smNPCs reached the 15th passage, the differentiation into midbrain neurons was induced. Here, we review To further validate the workflow and to also include a highly methylated sample, we performed Nanopore sequencing of an 897 bp synthetic methylated and unmethylated control DNA sample at different dilution ratios (0%, 50% and 100%). r, Spearman's rank correlation coefficient; p, Spearman's exploratory p-value. Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing. The plus-strand represents the L-strand and the minus-strand the H-strand. Revealing complete complex KIR haplotypes phased by long-read sequencing technology. 
This permits detecting expanded alleles outside of the range of normal PCR as well as interruption sequences (e.g., AGG units in the. to sequencing platforms. Mechanism of mitochondrial DNA replication in mouse L-cells: asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence.
Linearization of mtDNA has been recommended to counteract the influence of secondary and tertiary structure in bisulfite-dependent methods (Liu et al., 2016; Mechta et al., 2017; Owa et al., 2018). 
The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Oxford Nanopore MinION sequencing and genome assembly. Additionally, we used Megalodon, which resulted in low-level mtDNA CpG methylation as well (mean MF SD = 0.086 0.068). The mean coverage and MF of each data set were calculated. Similarly, when we used Megalodon, the detected MF was lower than expected in the 50% (mean MF SD = 0.040 0.024) and 100% (mean MF SD = 0.535 0.151) methylated sample as well (Supplementary Figure 6A). J. Pathol. illustrates sequencing molecule nanopore spectral The overall correlation between plus-strand and minus-strand methylation was stronger in the 45S rRNA gene compared to mtDNA (Spearman's r = 0.7011, Spearman's exploratory p-value < 0.0001, Figure 3B). doi: 10.1101/2021.02.05.429923, Mawlood, S. K., Dennany, L., Watson, N., Dempster, J., and Pickard, B. S. (2016). 31, 59705976. (2021). Copyright 2021 Lth, Wasner, Klein, Schaake, Tse, Pereira, La, Sinkkonen, Grnewald and Trinh. Rep. 6:23421. doi: 10.1038/srep23421, Lth, T., Klein, C., Schaake, S., Tse, R., Pereira, S., Lass, J., et al. The FPR was lower when the methylation was called with Nanopolish compared to Megalodon and there were fewer CpG sites that showed an MF > 0.2 in the amplicon sequencing data, indicating Nanopolish is less prone to false-positive calls in the context of mtDNA CpG methylation. This validation showed methylation profiles in the control and Parkin-PD patients comparable to the original analysis. Epigenetic clock: DNA methylation in aging. PLoS ONE 8:e59252.
an 897 bp synthetic DNA sample and the native 13 kb 45S rRNA nuclear-encoded gene. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. To investigate the methylation of the synthetic DNA, alignment, methylation calling, and calculation of MF were performed. Mapping DNA methylation with high-throughput nanopore sequencing. 
Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer. single dna detects nanopore nucleotide polymerase molecule device activity resolution 37, 973982. This includes disease-causing long Barcoding analysis of HIV drug resistance mutations using Oxford Nanopore MinION (ONT) sequencing.
We acknowledge Aurelien Ginolhac for his review of the manuscript and insightful comments and Philip Seibler for expertise in IPSC-derived neuronal models. Single-cell individual complete mtDNA sequencing uncovers hidden mitochondrial heterogeneity in human and mouse oocytes. But it is important to note that also with Nanopore sequencing we observed false-positive methylation calls as illustrated by the FPR calculated from the long-range PCR sequencing data. The most common method to detect mtDNA methylation has been bisulfite treatment. In addition, DNA was extracted from iPSC-derived neurons of four patients and three controls using the QIAAmp Minikit (Qiagen). These platforms require amplification of the template DNA for sequencing. Detection of GBA missense mutations and other variants using the Oxford Nanopore MinION. The control region of mitochondrial DNA shows an unusual CpG and non-CpG methylation pattern. 
Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers (A) Overall relationship between the mean coverage and mean methylation frequency of different subsections of the obtained sequencing data. In the D-loop, mtDNA replication and transcription are being initiated and there is evidence that methylation can interfere with these processes (Sirard, 2019). Despite our small sample size, we observed significant differences in patients with Parkin-PD, which highlights the importance to further investigate the underlying molecular mechanism and its implication in neurodegeneration. single sequencing molecule dna tsms sequencer reality making process figure americanlaboratory Rev. nanopore molecule coupled polymerases kinetics multiplex sequencing principle barcoded Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology. Nanopolish is based on HMM to classify each CpG site of each read as methylated or unmethylated (Simpson et al., 2017). Real-time DNA sequencing from single polymerase molecules. During the Guppy base-calling, the software reports Phred quality scores (qscores) of each read, which indicates the accuracy of the base-calling. The D5405 reference sequence provided by the manufacturer was used. On the one hand, there is evidence for a negative association between mtDNA methylation and gene expression (Sirard, 2019) and our results may indicate a higher expression of mtDNA genes in neuron-derived DNA. To evaluate the relationship between coverage and MF, we increased the coverage of each sample in a step-wise fashion and then called CpG methylation. 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). 
Please enter a term before submitting your search. Robust long-read native DNA sequencing using the ONT CsgG Nanopore system. doi: 10.1523/JNEUROSCI.4441-10.2011, Simpson, J. T., Workman, R. E., Zuzarte, P. C., David, M., Dursi, L. J., and Timp, W. (2017). Given the fact that, in our study, the overall methylation levels of samples are highly correlated between the tools, we used a global approach to assess differences in mtDNA CpG methylation between groups.
Rep. 11:8032. doi: 10.1038/s41598-021-87457-8, Guitton, R., Dlle, C., Alves, G., Tysnes, O.-B., Nido, G. S., and Tzoulis, C. (2021). The number of plus- and minus-strand reads from the mtDNA and 45S rRNA gene were comparable (Supplementary Figures 5C,D, Mann Whitney U-test p > 0.05). To validate if we obtained sufficient sequencing depth, we investigated the relationship between coverage and MF of mtDNA. Another limitation of this study is that Parkin mutation carriers are rare and an assessment of a larger Parkin-PD cohort is therefore only possible within the framework of international collaborations. DNA concentration of the blood- and neuron-derived DNA was quantified with Qubit fluorometric quantification using the dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). (C) Stacked bar plot of the fraction of reads that passed (blue) or failed (red) the Guppy quality threshold, stratified by the ratio of methylated reads in the sample. Genet.
To additionally investigate the frequency of homoplasmic and heteroplasmic variants in the mtDNA, we processed the indexed alignments in BAM format with the Mutserve pipeline (version 2.0.0) (Weissensteiner et al., 2016). bioRxiv [preprint]. Polymerase specific error rates and profiles identified by single molecule sequencing. (A) Methylation frequency detected by Nanopolish from three samples of synthetic DNA with different proportions of methylated DNA (0, 50 and 100%), only reads that passed Guppy quality threshold were included in the analysis. Detecting AGG interruptions in females with a FMR1 premutation by long-read single-molecule sequencing: a 1year clinical experience. 47, 1007210085. This significant difference was also present when we included a second independent batch of neuron-derived mtDNA (Supplementary Figure 8B). Real-time, portable genome sequencing for Ebola surveillance. Cell Biol. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. doi: 10.1371/journal.pone.0192722, Patil, V., Cuenin, C., Chung, F., Aguilera, J. R. R., Fernandez-Jimenez, N., Romero-Garmendia, I., et al. 
bioRxiv [preprint]. AgIn: measuring the landscape of CpG methylation of individual repetitive elements. We obtained a mean of 14 Mb data and a read length of 963 bp. The low levels of methylation have led to suggestions of this being artifactual, suggesting an absence of methylation in the mitochondrial genome altogether (Hong et al., 2013; Mechta et al., 2017). Thus, mtDNA methylation, although lower in neuron-derived mtDNA, appear to be conserved across different cell types. Library preparation of 1.5 g input DNA from the samples was performed with the Ligation Sequencing Kit (SQK-LSK109) (Oxford Nanopore Technologies, Oxford, United Kingdom) following a Nanopore whole-genome sequencing protocol (premium WGA). As previous studies have shown that there is non-CpG methylation present in the mtDNA (Dou et al., 2019; Patil et al., 2019), future workflows should be expanded to include non-CpG methylation as well. We observed a positive correlation between the plus- and minus-strand MF detected with Nanopolish (Spearman's r = 0.3214, Spearman's exploratory p-value < 0.0001, Figure 3A). sequencing nanopore adapted smrt diseases genetic The methylation frequency (MF) was calculated with the default threshold of the log-likelihood ratio. doi: 10.1128/MCB.00220-13, Jiang, S., and Guo, Y. doi: 10.1371/annotation/6a917a2e-df4a-4ad9-99bb-6aa7218b833e, Seibler, P., Graziotto, J., Jeong, H., Simunovic, F., Klein, C., and Krainc, D. (2011). Sci. Nucleic Acids Res. sequencing of single DNA (or RNA) molecules that does not require amplification during or before the sequencing process. Commun. Mov. The detected MF after the filtering changed marginally and was strongly correlated to the MF without this filtering step (Spearman's r = 0.9445, Spearman's exploratory p-value < 0.0001, Supplementary Figure 4). Rep. 10:7885. doi: 10.1038/s41598-020-64664-3, PubMed Abstract | CrossRef Full Text | Google Scholar, Bellizzi, D., D'aquila, P., Scafone, T., Giordano, M., Riso, V., Riccio, A., et al. To read this article in full you will need to make a payment. In the 0% methylated sample, a mean MF = 0.021 (SD 0.042) was detected similar to the 50% methylated sample (mean MF SD = 0.022 0.038).
Similarly, when we used Megalodon, the detected MF was higher in the 0% (mean MF SD = 0.054 0.070), 50% (mean MF SD = 0.645 0.168) and 100% methylated sample (mean MF SD = 0.687 0.182) (Supplementary Figure 6B). Electrophoresis 40, 272280. Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing. Although the correlation between plus- and minus-strand MF was higher in the 45S rRNA gene, the trend for strand differences in the mtDNA mean MF were minor and without bias toward any specific strand. doi: 10.18632/aging.100892, Mechta, M., Ingerslev, L. R., Fabre, O., Picard, M., and Barres, R. (2017). J. Hum. Mutations in Parkin, essential for degradation of damaged mitochondria, cause early-onset PD. Furthermore, due to the high cell culture costs, time and labor-intensive procedure, such mechanistic studies in iPSC-derived cell models are typically limited to a small number of samples. Nat. short for triplet (repeat) primed PCR, a PCR method for tandem-repeat analyses in which a third primer targeting the repeat is used. We obtained a mean coverage of 263X (SD = 79X) of the mitochondrial genome, which was on average 13X more coverage compared to just including the passed reads. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. doi: 10.1101/gr.234021.117, Georgieva, D., Liu, Q., Wang, K., and Egli, D. (2020). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi.2021.713084/full#supplementary-material, Aminuddin, A., Ng, P. Y., Leong, C. O., and Chua, E. W. (2020). We included patients with Parkin-PD and healthy control subjects and compared the mtDNA CpG methylation between the groups in a global fashion. Herein, we focus on the exploration of mtDNA CpG methylation using Nanopore. This methodology is useful when investigating the molecular underpinnings of neurodegenerative diseases. There was a weak correlation between the MF of individual CpG sites detected with Nanopolish and Megalodon (Spearman's r = 0.2924, Spearman's exploratory p-value < 0.0001, Supplementary Figure 2A) but a strong correlation between the mean MF of each sample (Spearman's r = 0.7904, Spearman's exploratory p-value = 0.0244, Supplementary Figure 2B). By contrast, with Megalodon there remained 28 unique CpG sites in the following mitochondrial genes: 12S rRNA (chrM:807, 897, 1022, 1132, 1176, 1215, 1225, 1487), 16S rRNA (chrM: 2565, 2569, 2571, 2842, 2914, 3077), NADH dehydrogenase, subunit 1 (chrM: 3405), tRNA-Methionine (chrM:4425), NADH dehydrogenase, subunit 2 (chrM: 4711), Cytochrome c oxidase, subunit 1 (chrM: 6688), NADH dehydrogenase, subunit 4 (chrM: 11715), NADH dehydrogenase, subunit 5 (chrM: 12816, 13364), tRNA-Glutamic acid (chrM:14683), Cytochrome b (chrM: 15039, 15589, 15759), tRNA-Threonine (chrM:15929) and within the non-coding D-loop (chrM:16410). Examining sources of error in PCR by single-molecule sequencing.
Utility and implications of exome sequencing in early-onset Parkinson's disease. Next, we explored possible NUMTs contamination in our data. or difficult to analyze by short-read platforms. to pathogenic microorganisms and viruses, constitutional disorders, pharmacogenomics, Using long-read, single-molecule sequencing technologies like Nanopore sequencing (Deamer et al., 2016), it is possible to overcome limitations of the indirect measurement of methylation with bisulfite treatment, as it can be detected directly from squiggle signals (Xu and Seki, 2020). 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. Am. 8:166. doi: 10.3389/fgene.2017.00166, Owa, C., Poulin, M., Yan, L. Y., and Shioda, T. (2018). (B) Overall relationship between the coverage of each individual CpG site and the corresponding methylation frequency. The DNA concentration was quantified as described above and 1 g input DNA was used. Thus, how mtDNA methylation affects mtDNA gene expression has not been fully elucidated yet. The other three are healthy control subjects without Parkin mutations (L-2131, L-2132 and L-2135). Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing. By contrast, there has also been supportive evidence for the presence of mtDNA methylation after linearization (Sun et al., 2018; Patil et al., 2019). To access possible false-positive methylation calls, we adjusted the MF for the FPR calculated from the methylation calling of the long-range PCR product (FPRNanopolish = 0.029, FPRMegalodon = 0.046) and excluded all CpG sites that showed an MF higher than 0.2 in the long-range PCR product from further analysis. 
To further validate the workflow, two types of commercially available 897 bp synthetic DNAs (D4505, Zymo Research, Irvine, CA, USA) were used where all cytosines were modified to 5-mC or unmodified. nanopore precise nucleotides sequencer labcritics pnas
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