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

Lee, Isac; Razaghi, Roham; Gilpatrick, Timothy; Molnar, Michael M.; Gershman, Ariel; Sadowski, Norah; Sedlazeck, Fritz J.; Hansen, Kasper D.; Simpson, Jared T.; Timp, Winston

doi:10.1038/s41592-020-01000-7

Cited by 166 publications

(191 citation statements)

References 46 publications

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“…With our nanoNOMe data accurately aligned to this region, we measured both chromatin accessibility and methylation, using reads greater than 20 kb to achieve a sequencing depth of 40X ( Fig S15f ). In this method, we use M.CviPI methyltransferase to decorate accessible chromatin with exogenous GpC methylation ( 17 ); using GpC sites we can then measure chromatin accessibility along with CpG methylation of the HG002 sequencing reads aligned to the HG002 chromosome X centromere assembly. Examining methylation we clearly observe a CDR in the HG002 chromosome X HOR region ( Fig 4a,b) .…”

Section: Resultsmentioning

confidence: 99%

Epigenetic Patterns in a Complete Human Genome

Gershman

Sauria

et al. 2021

Preprint

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The completion of the first telomere-to-telomere human genome, T2T-CHM13, enables exploration of the full epigenome, removing limitations previously imposed by the missing reference sequence. Existing epigenetic studies omit unassembled and unmappable genomic regions (e.g. centromeres, pericentromeres, acrocentric chromosome arms, subtelomeres, segmental duplications, tandem repeats). Leveraging the new assembly, we were able to measure enrichment of epigenetic marks with short reads using k-mer assisted mapping methods. This granted array-level enrichment information to characterize the epigenetic regulation of these satellite repeats. Using nanopore sequencing data, we generated base level maps of the most complete human methylome ever produced. We examined methylation patterns in satellite DNA and revealed organized patterns of methylation along individual molecules. When exploring the centromeric epigenome, we discovered a distinctive dip in centromere methylation consistent with active sites of kinetochore assembly. Through long-read chromatin accessibility measurements (nanoNOMe) paired to CUT&RUN data, we found the hypomethylated region was extremely inaccessible and paired to CENP-A/B binding. With long-reads we interrogated allele-specific, long-range epigenetic patterns in complex macro-satellite arrays such as those involved in X chromosome inactivation. Using the single molecule measurements we can clustered reads based on methylation status alone distinguishing epigenetically heterogeneous and homogeneous areas. The analysis provides a framework to investigate the most elusive regions of the human genome, applying both long and short-read technology to grant new insights into epigenetic regulation.

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Section: Resultsmentioning

confidence: 99%

Epigenetic Patterns in a Complete Human Genome

Gershman

Sauria

et al. 2021

Preprint

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“…The methylation caller generates a log-likelihood value for the ratio of probability of methylated to unmethylated CpGs at a specific k -mer. We filtered methylation calls using the nanopore_methylation_utilities tool ( https://github.com/isaclee/nanopore-methylation-utilities ) 53 , which uses a log-likelihood ratio of 2.5 as a threshold for calling methylation. 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: Methodsmentioning

confidence: 99%

The structure, function and evolution of a complete human chromosome 8

Logsdon

Vollger

Hsieh

et al. 2021

Nature

273

278

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The complete assembly of each human chromosome is essential for understanding human biology and evolution1,2. Here we use complementary long-read sequencing technologies to complete the linear assembly of human chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08-Mb centromeric α-satellite array, a 644-kb copy number polymorphism in the β-defensin gene cluster that is important for disease risk, and an 863-kb variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric α-satellite array is generally methylated except for a 73-kb hypomethylated region of diverse higher-order α-satellites enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. In addition, we confirm the overall organization and methylation pattern of the centromere in a diploid human genome. Using a dual long-read sequencing approach, we complete high-quality draft assemblies of the orthologous centromere from chromosome 8 in chimpanzee, orangutan and macaque to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order α-satellite structure evolved in the great ape ancestor with a layered symmetry, in which more ancient higher-order repeats locate peripherally to monomeric α-satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated by more than 2.2-fold compared to the unique portions of the genome, and this acceleration extends into the flanking sequence.

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“…Prerequisite for these studies will be the generation of single molecule nucleosome configuration data as in Brown et al, 2013 at different time points of chromatin opening. It should be noted that such data are nowadays much easier to obtain than by the psoralen-TEM approach of Brown et al, 2013 due to the recent development of single molecule long-read sequencing of DNA methylation footprints ( Shipony et al, 2020 ; Stergachis et al, 2020 ; Lee et al, 2020 ; Oberbeckmann et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

“…A second line of studies may investigate the opening and closing dynamics in the wild-type scenario in more detail. We argued that the experimentally observed faster closing than opening is consistent with regulation by assembly and inconsistent with regulation by disassembly (see Stergachis et al, 2020;Lee et al, 2020;Oberbeckmann et al, 2019).…”

Section: Experimentally Testable Predictionsmentioning

confidence: 93%

Effective dynamics of nucleosome configurations at the yeast PHO5 promoter

Wolff

Schmid

Korber

et al. 2021

eLife

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Chromatin dynamics are mediated by remodeling enzymes and play crucial roles in gene regulation, as established in a paradigmatic model, the S. cerevisiae PHO5 promoter. However, effective nucleosome dynamics, i.e. trajectories of promoter nucleosome configurations, remain elusive. Here, we infer such dynamics from the integration of published single-molecule data capturing multi-nucleosome configurations for repressed to fully active PHO5 promoter states with other existing histone turnover and new chromatin accessibility data. We devised and systematically investigated a new class of 'regulated on-off-slide' models simulating global and local nucleosome (dis)assembly and sliding. Only seven of 68145 models agreed well with all data. All seven models involve sliding and the known central role of the N-2 nucleosome, but regulate promoter state transitions by modulating just one assembly rather than disassembly process. This is consistent with but challenges common interpretations of previous observations at the PHO5 promoter and suggests chromatin opening by binding competition.

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

Cited by 166 publications

References 46 publications

Epigenetic Patterns in a Complete Human Genome

Epigenetic Patterns in a Complete Human Genome

The structure, function and evolution of a complete human chromosome 8

Effective dynamics of nucleosome configurations at the yeast PHO5 promoter

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