Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

Lee, Isac; Razaghi, Roham; Gilpatrick, Timothy; Sadowski, Norah; Sedlazeck, Fritz J.; Timp, Winston

doi:10.1101/504993

Cited by 26 publications

(32 citation statements)

References 29 publications

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“…[67]. nanoNOMe, MeSMLR-seq, and SMAC-seq are methods that combine the accessibility of one or more methyltransferases and the direct detection of methylated regions by the Nanopore sequencer instead of requiring BS-seq [68][69][70]. These methods can detect open chromatin patterns of single long DNA molecules (Fig.…”

Section: Tools For Modified Base Detectionmentioning

confidence: 99%

“…In SMAC-seq, Tombo is used for methylation detection [68]. Lee et al applied nanoNOMe to four human cell lines and detected DNA methylation using Nanopolish [69]. Furthermore, they investigated not only open chromatin patterns but also CpG methylation from single reads and showed an anticorrelation between chromatin accessibility and DNA methylation.…”

Section: Tools For Modified Base Detectionmentioning

confidence: 99%

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Recent advances in the detection of base modifications using the Nanopore sequencer

Seki

2019

J Hum Genet

107

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DNA and RNA modifications have important functions, including the regulation of gene expression. Existing methods based on short-read sequencing for the detection of modifications show difficulty in determining the modification patterns of single chromosomes or an entire transcript sequence. Furthermore, the kinds of modifications for which detection methods are available are very limited. The Nanopore sequencer is a single-molecule, long-read sequencer that can directly sequence RNA as well as DNA. Moreover, the Nanopore sequencer detects modifications on long DNA and RNA molecules. In this review, we mainly focus on base modification detection in the DNA and RNA of mammals using the Nanopore sequencer. We summarize current studies of modifications using the Nanopore sequencer, detection tools using statistical tests or machine learning, and applications of this technology, such as analyses of open chromatin, DNA replication, and RNA metabolism.

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Section: Tools For Modified Base Detectionmentioning

confidence: 99%

Section: Tools For Modified Base Detectionmentioning

confidence: 99%

Recent advances in the detection of base modifications using the Nanopore sequencer

Seki

2019

J Hum Genet

107

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show abstract

“…The methylation caller generates a log-likelihood value for the ratio of probability of methylated to unmethylated CGs at a specific k -mer. We next filtered methylation calls using the nanopore_methylation_utilities tool ( https://github.com/isaclee/nanopore-methylation-utilities ), which uses a log-likelihood ratio of 2.5 as a threshold for calling methylation 59 . CpG sites with log-likelihood ratios greater than 2.5 (methylated) or less than -2.5 (unmethylated) are considered high-quality and included in the analysis.…”

Section: Methylation Analysismentioning

confidence: 99%

Telomere-to-telomere assembly of a complete human X chromosome

Miga

Koren

Rhie

et al. 2019

Preprint

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After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2 . The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2 , along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the ~2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.Complete, telomere-to-telomere reference assemblies are necessary to ensure that all genomic variants, large and small, are discovered and studied. Currently, unresolved regions of the human genome are defined by multi-megabase satellite arrays in the pericentromeric regions and the rDNA arrays on acrocentric short arms, as well as regions enriched in segmental duplications that are greater than hundreds of kilobases in length and greater than 98% identical between paralogs. Due to their absence from the reference, these repeat-rich sequences are often excluded from contemporary genetics and genomics studies, limiting the scope of association and functional analyses 4,5 . Unresolved repeat sequences also result in unintended consequences such as paralogous sequence variants incorrectly called as allelic v ariants 6 and even the contamination of bacterial gene databases 7 . Completion of the entire human genome is expected to contribute to our understanding of chromosome function 8 and human disease 9 , and a comprehensive understanding of genomic variation will improve the driving technologies in biomedicine that currently use short-read mapping to a reference genome (e.g. RNA-seq 10 , ChIP-seq 11 , ATAC-seq 12 ).The fundamental challenge of reconstructing a genome from many comparatively short sequencing reads-a process known as genome assembly-is distinguishing the repeated sequences from one another 13 . Resolving such r...

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“…Not only the landscape of alternative splicing can be investigated by reading through entire isoforms [33], but the various base modifications present on native RNA molecules can also be detected using this PCR-free method [18]. Moreover, native CpG methylation and chromatin accessibility can be studied in parallel using long reads [38].…”

Section: Long-read Sequencing In Human Genetics Abstractmentioning

confidence: 99%

“…Methylation analysis [20,22,38] Microbiome analysis [35,46,55] Pseudogene discrimination CYP2D6 [40], IKBKG [4], PKD1 [5], SMN1…”

Section: Infectionmentioning

confidence: 99%

Long-read sequencing in human genetics

Kraft

Kurth

2019

Medizinische Genetik

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Sanger sequencing revolutionized molecular genetics 40 years ago. However, next-generation sequencing technologies became further game changers and shaped our current view on genome structure and function in health and disease. Although still at the very beginning, third-generation sequencing methods, also referred to as long-read sequencing technologies, provide exciting possibilities for studying structural variations, epigenetic modifications, or repetitive elements and complex regions of the genome. We discuss the advantages and pitfalls of current long-read sequencing methods with a focus on nanopore sequencing, summarize respective applications and provide an outlook on the potential of these novel methods.

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Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

Cited by 26 publications

References 29 publications

Recent advances in the detection of base modifications using the Nanopore sequencer

Recent advances in the detection of base modifications using the Nanopore sequencer

Telomere-to-telomere assembly of a complete human X chromosome

Long-read sequencing in human genetics

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