scholarly journals Genome-Wide Detection of Imprinted Differentially Methylated Regions Using Nanopore Sequencing

2021 ◽  
Author(s):  
Vahid Akbari ◽  
Jean-Michel Garant ◽  
Kieran O'Neill ◽  
Pawan Pandoh ◽  
Richard Moore ◽  
...  
Keyword(s):  
B Lymphocyte ◽  
Sequence Data ◽  
Nanopore Sequencing ◽  
Differentially Methylated Regions ◽  
Methylation Array ◽  
Current State ◽  
Genome Wide ◽  
Long Reads ◽  
Genome Bisulfite Sequencing ◽  
Potential Applications

Imprinting is a critical part of normal embryonic development in mammals, controlled by defined parent-of-origin (PofO) differentially methylated regions (DMRs) known as imprinting control regions. As we and others have shown, direct nanopore sequencing of DNA provides a mean to detect allelic methylation and to overcome the drawbacks of methylation array and short-read technologies. Here we leverage publicly-available nanopore sequence data for 12 standard B-lymphocyte cell lines to present the first genome-wide mapping of imprinted intervals in humans using this technology. We were able to phase 95% of the human methylome and detect 94% of the well-characterized imprinted DMRs. In addition, we found 28 novel imprinted DMRs (12 germline and 16 somatic), which we confirmed using whole-genome bisulfite sequencing (WGBS) data. Analysis of WGBS data in mouse, rhesus, and chimp suggested that 12 of these are conserved. We also detected subtle parental methylation bias spanning several kilobases at seven known imprinted clusters. These results expand the current state of knowledge of imprinting, with potential applications in the clinic. We have also demonstrated that nanopore long reads, can reveal imprinting using only parent-offspring trios, as opposed to the large multi-generational pedigrees that have previously been required.

scholarly journals Megabase-scale methylation phasing using nanopore long reads and NanoMethPhase

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Vahid Akbari ◽  
Jean-Michel Garant ◽  
Kieran O’Neill ◽  
Pawan Pandoh ◽  
Richard Moore ◽  
...  
Keyword(s):  
Sequence Data ◽  
Software Tool ◽  
Nanopore Sequencing ◽  
Post Process ◽  
Genome Wide ◽  
Long Reads ◽  
Improve Accuracy ◽  
Allele Specific ◽  
Allele Specific Methylation ◽  
Low Coverage

AbstractThe ability of nanopore sequencing to simultaneously detect modified nucleotides while producing long reads makes it ideal for detecting and phasing allele-specific methylation. However, there is currently no complete software for detecting SNPs, phasing haplotypes, and mapping methylation to these from nanopore sequence data. Here, we present NanoMethPhase, a software tool to phase 5-methylcytosine from nanopore sequencing. We also present SNVoter, which can post-process nanopore SNV calls to improve accuracy in low coverage regions. Together, these tools can accurately detect allele-specific methylation genome-wide using nanopore sequence data with low coverage of about ten-fold redundancy.

1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 126
Author(s):  
J. E. Duan ◽  
Z. Jiang ◽  
F. Alqahtani ◽  
I. Mandoiu ◽  
H. Dong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Epigenetic Regulation ◽  
Bisulfite Sequencing ◽  
Whole Genome ◽  
Differentially Methylated Regions ◽  
Whole Genome Bisulfite Sequencing ◽  
Cpg Sites ◽  
Genome Wide ◽  
Genome Bisulfite Sequencing

Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.

scholarly journals The SEQC2 Epigenomics Quality Control (EpiQC) Study: Comprehensive Characterization of Epigenetic Methods, Reproducibility, and Quantification

2020 ◽  
Author(s):  
Jonathan Foox ◽  
Jessica Nordlund ◽  
Claudia Lalancette ◽  
Ting Gong ◽  
Michelle Lacey ◽  
...  
Keyword(s):  
Quality Control ◽  
Bisulfite Sequencing ◽  
Sequence Data ◽  
Basic Research ◽  
High Coverage ◽  
Genome Wide ◽  
Wide Range ◽  
Genome Bisulfite Sequencing ◽  
Mammalian Genomes ◽  
Primary Focus

AbstractDetection of DNA cytosine modifications such as 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) is essential for understanding the epigenetic changes that guide development, cellular lineage specification, and disease. The wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research.We present a multi-platform assessment and a global resource for epigenetics research from the FDA’s Epigenomics Quality Control (EpiQC) Group. The study design leverages seven human cell lines that are publicly available from the National Institute of Standards and Technology (NIST) and Genome in a Bottle (GIAB) consortium. These genomes were subject to a variety of genome-wide methylation interrogation approaches across six independent laboratories. Our primary focus was on cytosine modifications found in mammalian genomes (5mC, 5hmC). Each sample was processed in two or more technical replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS, SPLAT), oxidative bisulfite sequencing (oxBS), Enzymatic Methyl-seq (EM-seq), Illumina EPIC targeted-methylation sequencing, and ATAC-seq. Each library was sequenced to high coverage on an Illumina NovaSeq 6000. The data were subject to rigorous quality assessment and subsequently compared to Illumina EPIC methylation microarrays. We provide a wide range of sequence data for commonly used genomics reference materials, as well as best practices for epigenomics research. These findings can serve as a guide for researchers to enable epigenomic analysis of cellular identity in development, health, and disease.

scholarly journals Nanopore sequencing and assembly of a human genome with ultra-long reads

2017 ◽  
Author(s):  
Miten Jain ◽  
S Koren ◽  
J Quick ◽  
AC Rand ◽  
TA Sasani ◽  
...  
Keyword(s):  
Human Genome ◽  
Cancer Progression ◽  
De Novo ◽  
Sequence Data ◽  
Point Of Care ◽  
Genetic Diseases ◽  
Nanopore Sequencing ◽  
Repeat Structure ◽  
Long Reads ◽  
Amazon Web Services

AbstractNanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation. However until recently nanopore sequencing has been mainly applied to small genomes, due to the limited output attainable. We present nanopore sequencing and assembly of the GM12878 Utah/Ceph human reference genome generated using the Oxford Nanopore MinION and R9.4 version chemistry. We generated 91.2 Gb of sequence data (∼30× theoretical coverage) from 39 flowcells. De novo assembly yielded a highly complete and contiguous assembly (NG50 ∼3Mb). We observed considerable variability in homopolymeric tract resolution between different basecallers. The data permitted sensitive detection of both large structural variants and epigenetic modifications. Further we developed a new approach exploiting the long-read capability of this system and found that adding an additional 5×-coverage of ‘ultra-long’ reads (read N50 of 99.7kb) more than doubled the assembly contiguity. Modelling the repeat structure of the human genome predicts extraordinarily contiguous assemblies may be possible using nanopore reads alone. Portable de novo sequencing of human genomes may be important for rapid point-of-care diagnosis of rare genetic diseases and cancer, and monitoring of cancer progression. The complete dataset including raw signal is available as an Amazon Web Services Open Dataset at: https://github.com/nanopore-wgs-consortium/NA12878.

scholarly journals Long read sequencing of 3,622 Icelanders provides insight into the role of structural variants in human diseases and other traits

2019 ◽  
Author(s):  
Doruk Beyter ◽  
Helga Ingimundardottir ◽  
Asmundur Oddsson ◽  
Hannes P. Eggertsson ◽  
Eythor Bjornsson ◽  
...  
Keyword(s):  
Sequence Data ◽  
P Value ◽  
Repeat Region ◽  
Structural Variants ◽  
Genome Wide ◽  
Population Average ◽  
Long Reads ◽  
Oxford Nanopore ◽  
A Genome ◽  
Long Read

Long-read sequencing (LRS) promises to improve characterization of structural variants (SVs), a major source of genetic diversity. We generated LRS data on 3,622 Icelanders using Oxford Nanopore Technologies, and identified a median of 22,636 SVs per individual (a median of 13,353 insertions and 9,474 deletions), spanning a median of 10 Mb per haploid genome. We discovered a set of 133,886 reliably genotyped SV alleles and imputed them into 166,281 individuals to explore their effects on diseases and other traits. We discovered an association with a rare (AF = 0.037%) deletion of the first exon of PCSK9. Carriers of this deletion have 0.93 mmol/L (1.31 SD) lower LDL cholesterol levels than the population average (p-value = 7.0·10−20). We also discovered an association with a multi-allelic SV inside a large repeat region, contained within single long reads, in an exon of ACAN. Within this repeat region we found 11 alleles that differ in the number of a 57 bp-motif repeat, and observed a linear relationship (0.016 SD per motif inserted, p = 6.2·10−18) between the number of repeats carried and height. These results show that SVs can be accurately characterized at population scale using long read sequence data in a genome-wide non-targeted approach and demonstrate how SVs impact phenotypes.

scholarly journals Genome-wide search of nucleosome patterns using visual analytics

2018 ◽  
Vol 35 (13) ◽  
pp. 2185-2192
Author(s):  
Rodrigo Santamaría ◽  
Roberto Therón ◽  
Laura Durán ◽  
Alicia García ◽  
Sara González ◽  
...  
Keyword(s):  
Visual Analytics ◽  
Sequence Data ◽  
Pattern Search ◽  
Supplementary Information ◽  
Visual Discourse ◽  
Genome Wide ◽  
Genome Wide Search ◽  
Potential Applications ◽  
Burrows Wheeler Transform ◽  
Different Sources

Abstract Motivation The Burrows-Wheeler transform (BWT) is widely used for the fast alignment of high-throughput sequence data. This method also has potential applications in other areas of bioinformatics, and it can be specially useful for the fast searching of patterns on coverage data from different sources. Results We present a nucleosome pattern search method that converts levels of nucleosomal occupancy to a sequence-like format to which BWT searches can be applied. The method is embedded in a nucleosome map browser, ‘Nucleosee‘, an interactive visual tool specifically designed to enhance BWT searches, giving them context and making them suitable for visual discourse analysis of the results. The proposed method is fast, flexible and sufficiently generic for the exploration of data in a broad and interactive way. Availability and implementation The proposed algorithm and visual browser are available for testing at http://cpg3.der.usal.es/nucleosee. The source code and installation packages are also available at https://github.com/rodrigoSantamaria/nucleosee. Supplementary information Supplementary data are available at Bioinformatics online.

Short Review and opinion: New matter properties and applications based on Hybrids Graphene based Metamaterials

2020 ◽  
Vol 04 ◽  
Author(s):  
A. Guillermo Bracamonte
Keyword(s):  
Short Review ◽  
New Materials ◽  
Design And Synthesis ◽  
Chemical Structures ◽  
Current State ◽  
Potential Applications ◽  
Fundamental Research ◽  
Miniaturized Instrumentation ◽  
And Control ◽  
Conductive Properties

: Graphene as Organic material showed special attention due to their electronic and conductive properties. Moreover, its highly conjugated chemical structures and relative easy modification permitted varied design and control of targeted properties and applications. In addition, this Nanomaterial accompanied with pseudo Electromagnetic fields permitted photonics, electronics and Quantum interactions with their surrounding that generated new materials properties. In this context, this short Review, intends to discuss many of these studies related with new materials based on graphene for light and electronic interactions, conductions, and new modes of non-classical light generation. It should be highlighted that these new materials and metamaterials are currently in progress. For this reason it was showed and discussed some representative examples from Fundamental Research with Potential Applications as well as for their incorporations to real Advanced devices and miniaturized instrumentation. In this way, it was proposed this Special issue entitled “Design and synthesis of Hybrids Graphene based Metamaterials”, in order to open and share the knowledge of the Current State of the Art in this Multidisciplinary field.

scholarly journals Dynamic Manipulation of THz Waves Enabled by Phase-Transition VO2 Thin Film

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 114
Author(s):  
Chang Lu ◽  
Qingjian Lu ◽  
Min Gao ◽  
Yuan Lin
Keyword(s):  
Optical Methods ◽  
Stimuli Responsive ◽  
Device Structure ◽  
Current State ◽  
Insulator Metal Transition ◽  
Potential Applications ◽  
Fs Laser ◽  
External Stimuli ◽  
Thz Applications ◽  
Thz Waves

The reversible and multi-stimuli responsive insulator-metal transition of VO2, which enables dynamic modulation over the terahertz (THz) regime, has attracted plenty of attention for its potential applications in versatile active THz devices. Moreover, the investigation into the growth mechanism of VO2 films has led to improved film processing, more capable modulation and enhanced device compatibility into diverse THz applications. THz devices with VO2 as the key components exhibit remarkable response to external stimuli, which is not only applicable in THz modulators but also in rewritable optical memories by virtue of the intrinsic hysteresis behaviour of VO2. Depending on the predesigned device structure, the insulator-metal transition (IMT) of VO2 component can be controlled through thermal, electrical or optical methods. Recent research has paid special attention to the ultrafast modulation phenomenon observed in the photoinduced IMT, enabled by an intense femtosecond laser (fs laser) which supports “quasi-simultaneous” IMT within 1 ps. This progress report reviews the current state of the field, focusing on the material nature that gives rise to the modulation-allowed IMT for THz applications. An overview is presented of numerous IMT stimuli approaches with special emphasis on the underlying physical mechanisms. Subsequently, active manipulation of THz waves through pure VO2 film and VO2 hybrid metamaterials is surveyed, highlighting that VO2 can provide active modulation for a wide variety of applications. Finally, the common characteristics and future development directions of VO2-based tuneable THz devices are discussed.

scholarly journals In vivo interactome profiling by enzyme‐catalyzed proximity labeling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yangfan Xu ◽  
Xianqun Fan ◽  
Yang Hu
Keyword(s):  
State Of The Art ◽  
Catalytic Efficiency ◽  
Biological Processes ◽  
Protein Protein Interaction ◽  
Current State ◽  
Protein Interactome ◽  
Potential Applications ◽  
Protein Protein Interaction Networks ◽  
Temporal And Spatial

AbstractEnzyme-catalyzed proximity labeling (PL) combined with mass spectrometry (MS) has emerged as a revolutionary approach to reveal the protein-protein interaction networks, dissect complex biological processes, and characterize the subcellular proteome in a more physiological setting than before. The enzymatic tags are being upgraded to improve temporal and spatial resolution and obtain faster catalytic dynamics and higher catalytic efficiency. In vivo application of PL integrated with other state of the art techniques has recently been adapted in live animals and plants, allowing questions to be addressed that were previously inaccessible. It is timely to summarize the current state of PL-dependent interactome studies and their potential applications. We will focus on in vivo uses of newer versions of PL and highlight critical considerations for successful in vivo PL experiments that will provide novel insights into the protein interactome in the context of human diseases.

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