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

Liu, Yang; Rosikiewicz, Wojciech; Pan, Zikang; Jillette, Nathaniel; Wang, Ping; Taghbalout, Aziz; Foox, Jonathan; Mason, Christopher E.; Carroll, Martin; Cheng, Albert W.; Li, Sheng

doi:10.1101/2021.05.05.442849

Cited by 8 publications

(6 citation statements)

References 83 publications

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“…Here we represent the first comprehensive comparison of computational tools focused on detecting m6A in the ONT DRS dataset. Rational oligo models and well-established BS-seq data have already been applied to the benchmarking of 5mC detection by nanopore sequencing 44,45 . However, there is no gold-standard method for m6A detection with both single-base resolution and stoichiometric information on the NGS platform.…”

Section: Discussionmentioning

confidence: 99%

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing

et al. 2023

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N6-methyladenosine (m6A) has been increasingly recognized as a new and important regulator of gene expression. To date, transcriptome-wide m6A detection primarily relies on well-established methods using next-generation sequencing (NGS) platform. However, direct RNA sequencing (DRS) using the Oxford Nanopore Technologies (ONT) platform has recently emerged as a promising alternative method to study m6A. While multiple computational tools are being developed to facilitate the direct detection of nucleotide modifications, little is known about the capabilities and limitations of these tools. Here, we systematically compare ten tools used for mapping m6A from ONT DRS data. We find that most tools present a trade-off between precision and recall, and integrating results from multiple tools greatly improve performance. Using a negative control could improve precision by subtracting certain intrinsic bias. We also observed variation in detection capabilities and quantitative information among motifs, and identified sequencing depth and m6A stoichiometry as potential factors affecting performance. Our study provides insight into the computational tools currently used for mapping m6A based on ONT DRS data and highlights the potential for further improving these tools, which may serve as the basis for future research.

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Section: Discussionmentioning

confidence: 99%

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing

et al. 2023

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“…Calling DNAm from Nanopore sequencing data is a computational challenge to classify methylated and unmethylated cytosines based on the electrical signal emitted as the DNA passes through the pore with a number of algorithms developed for this purpose. In this study we implemented just one of these algorithms, Nanopolish(4), which was found to be consistently one of the most accurate and concordant with the other best performing methods, across a range of genomic contexts, as well as the least computationally intensive (21). One limitation of this algorithm is that it only considers CpGs and ignores DNAm at cytosines in other genomic contexts.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of nanopore sequencing for epigenetic epidemiology: a comparison with DNA methylation microarrays

Flynn

Washer

Jeffries

et al. 2022

Preprint

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Most epigenetic epidemiology to date has utilized microarrays to identify positions in the genome where variation in DNA methylation is associated with environmental exposures or disease. However, these profile less than 3% of DNA methylation sites in the human genome, potentially missing affected loci and preventing the discovery of disrupted biological pathways. Third generation sequencing technologies, including Nanopore sequencing, have the potential to revolutionise the generation of epigenetic data, not only by providing genuine genome-wide coverage but profiling epigenetic modifications direct from native DNA. Here we assess the viability of using Nanopore sequencing for epidemiology by performing a comparison with DNA methylation quantified using the most comprehensive microarray available, the Illumina EPIC array. We implemented a CRISPR-Cas9 targeted sequencing approach in concert with Nanopore sequencing to profile DNA methylation in three genomic regions to attempt to rediscover genomic positions that existing technologies have shown are differentially methylated in tobacco smokers. Using Nanopore sequencing reads, DNA methylation was quantified at 1,779 CpGs across three regions, providing a finer resolution of DNA methylation patterns compared to the EPIC array. The correlation of estimated levels of DNA methylation between platforms was high. Furthermore, we identified 12 CpGs where hypomethylation was significantly associated with smoking status, including 10 within the AHRR gene. In summary, Nanopore sequencing is a valid option for identifying genomic loci where large differences in DNAm are associated with a phenotype and has the potential to advance our understanding of the role differential methylation plays in the aetiology of complex disease.

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“…In real-time single molecule sequencing approaches, methylation impacts the kinetics of the polymerase while incorporating fluorescently labeled nucleotides into nucleic acid strands during DNA sequencing: base modifications alter both pulse width and interpulse duration, allowing the detection of epigenetics changes (Flusberg et al, 2010). In nanoporebased technologies, the electric current pattern is different for modified and non-modified bases and methylation state can be predicted with specific tools (Liu et al, 2021).…”

Section: Application Of Lrs To the Diagnosis Of Imprinting Disordersmentioning

confidence: 99%

Long read sequencing on its way to the routine diagnostics of genetic diseases

Olivucci,

Iovino,

Innella

et al. 2024

Front. Genet.

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The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.

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

Cited by 8 publications

References 83 publications

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing

Evaluation of nanopore sequencing for epigenetic epidemiology: a comparison with DNA methylation microarrays

Long read sequencing on its way to the routine diagnostics of genetic diseases

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