scholarly journals Targeted Gene Bisulfite Sequencing Identifies Differential Methylation in p21

2018 ◽  
Author(s):  
Ramya T. Kolli ◽  
Travis C. Glenn ◽  
Bradley T. Brown ◽  
Lillie M. Barnett ◽  
Lawrence H. Lash ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Bisulfite Sequencing ◽  
Kinase Inhibitor ◽  
Rat Kidney ◽  
Low Cost ◽  
Illumina Miseq ◽  
Kidney Cells ◽  
Human Embryonic Kidney Cells ◽  
Reduced Representation

AbstractNext-generation sequencing (NGS) methods are widely available to assess methylation of whole-genomes, reduced representation of genomes, and target capture of many loci, but simple, flexible, and low-cost methods are needed to leverage NGS for sequencing single-locus amplicons from large numbers of samples. We developed a two-stage PCR approach, targeted gene bisulfite sequencing (TGBS) which uses the Illumina MiSeq and Bismark bisulfite mapper, to assess site specific changes in methylation of the cyclin-dependent kinase inhibitor p21 (CDKN1a) after exposure to a DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine (5-Aza) and determine the differences between human and rat p21 methylation. TGBS analysis of human embryonic kidney cells (HEK293) and human proximal tubular cells (hPT) demonstrated variation at a known methylation sensitive site (SIE-1), but not in rat kidney cells. Treatment of cells with 5-Aza altered the methylation of this site in correlation with increased p21 protein expression. We also found that human and rat p21 promoter sequences differ considerably in the amount of basal DNA methylation. These data showed the utility of TGBS for rapid analysis of DNA methylation of specific loci. We provide links to a ready-to-run Virtualbox that includes the program and commands for methylation analysis of bisulfite datasets, including step-by-step directions.

scholarly journals How to Design a Whole-Genome Bisulfite Sequencing Experiment

Epigenomes ◽  
2018 ◽  
Vol 2 (4) ◽  
pp. 21 ◽  
Author(s):  
Claudius Grehl ◽  
Markus Kuhlmann ◽  
Claude Becker ◽  
Bruno Glaser ◽  
Ivo Grosse
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Alternative Methods ◽  
Transcriptional Gene Silencing ◽  
Whole Genome ◽  
Reduced Representation ◽  
Genome Region ◽  
Whole Genome Bisulfite Sequencing ◽  
Sequencing Experiment ◽  
Genome Bisulfite Sequencing

Aside from post-translational histone modifications and small RNA populations, the epigenome of an organism is defined by the level and spectrum of DNA methylation. Methyl groups can be covalently bound to the carbon-5 of cytosines or the carbon-6 of adenine bases. DNA methylation can be found in both prokaryotes and eukaryotes. In the latter, dynamic variation is shown across species, along development, and by cell type. DNA methylation usually leads to a lower binding affinity of DNA-interacting proteins and often results in a lower expression rate of the subsequent genome region, a process also referred to as transcriptional gene silencing. We give an overview of the current state of research facilitating the planning and implementation of whole-genome bisulfite-sequencing (WGBS) experiments. We refrain from discussing alternative methods for DNA methylation analysis, such as reduced representation bisulfite sequencing (rrBS) and methylated DNA immunoprecipitation sequencing (MeDIPSeq), which have value in specific experimental contexts but are generally disadvantageous compared to WGBS.

Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

2011 ◽  
Vol 6 (4) ◽  
pp. 468-481 ◽  
Author(s):  
Hongcang Gu ◽  
Zachary D Smith ◽  
Christoph Bock ◽  
Patrick Boyle ◽  
Andreas Gnirke ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Reduced Representation ◽  
Reduced Representation Bisulfite Sequencing ◽  
Dna Methylation Profiling ◽  
Genome Scale ◽  
Methylation Profiling

scholarly journals DNA methylation estimation using methylation-sensitive restriction enzyme bisulfite sequencing (MREBS)

2017 ◽  
Author(s):  
Giancarlo Bonora ◽  
Liudmilla Rubbi ◽  
Marco Morselli ◽  
Constantinos Chronis ◽  
Kathrin Plath ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Restriction Enzyme ◽  
Large Scale ◽  
Bisulfite Sequencing ◽  
Multiple Regression Model ◽  
Reduced Representation ◽  
A Genome ◽  
Genome Bisulfite Sequencing ◽  
Methylation Sensitive Restriction Enzyme ◽  
Wide Scale

ABSTRACTWhole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) are widely used for measuring DNA methylation levels on a genome-wide scale(1). Both methods have limitations: WGBS is expensive and prohibitive for most large-scale projects; RRBS only interrogates 6-12% of the CpGs in the human genome(16,19). Here, we introduce methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) which has the reduced sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. We built a multiple regression model that combines the two features of MREBS: the bisulfite conversion ratios of single cytosines (as in WGBS and RRBS) as well as the number of reads that cover each locus (as in MRE-seq(12)). This combined approach allowed us to estimate differential methylation across 60% of the genome using read count data alone, and where counts were sufficiently high in both samples (about 1.5% of the genome), our estimates were significantly improved by the single CpG conversion information. We show that differential DNA methylation values based on MREBS data correlate well with those based on WGBS and RRBS. This newly developed technique combines the sequencing cost of RRBS and DNA methylation estimates on a portion of the genome similar to WGBS, making it ideal for large-scale projects of mammalian genomes.

scholarly journals DNA methylation in cockroaches is essential in early embryo development and reduces gene expression noise

2020 ◽  
Author(s):  
Alba Ventos-Alfonso ◽  
Guillem Ylla ◽  
Jose-Carlos Montañes ◽  
Xavier Belles
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Early Embryo ◽  
Expression Change ◽  
Reduced Representation ◽  
Gene Expression Noise ◽  
Expression Variability ◽  
Functional Studies ◽  
Expression Noise ◽  
Hypermethylated Genes

AbstractThe influence of DNA methylation on gene behavior, and its consequent phenotypic effects appear to be very important, but the details are not well understood. Insects offer a diversity of DNA methylation modes, making them an excellent lineage for comparative analyses. However, functional studies have tended to focus on quite specialized holometabolan species, such as wasps, bees, beetles, and flies. Here we have studied DNA methylation in a hemimetabolan insect, the cockroach Blattella germanica, a model of early-branching insects. In this cockroach, one of the main genes responsible for DNA methylation, DNA methyltransferase 1 (DNMT1), is expressed in early embryogenesis. In our experiments, DNMT1 interference by RNAi reduces DNA methylation and impairs blastoderm formation. Using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic analyses, we observed that hypermethylated genes are associated with metabolism and are highly expressed, whereas hypomethylated genes are related to signaling and have low expression levels. Moreover, the expression change in hypermethylated genes is greter than that in hypomethylated genes, whereas hypermethylated genes have less expression variability than hypomethylated genes. The latter observation has also been reported for humans and in Arabidopsis plants. A reduction in expression noise may therefore be one of the few universal effects of DNA methylation.

scholarly journals GENE-40. CHARACTERIZING EPIGENETIC INTRATUMORAL HETEROGENEITY IN GLIOMA USING SINGLE-CELL BISULFITE SEQUENCING

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi106-vi106
Author(s):  
Kevin C Johnson ◽  
Kevin Anderson ◽  
Elise Courtois ◽  
Floris Barthel ◽  
Michael Samuels ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Bisulfite Sequencing ◽  
Cpg Island ◽  
Regulatory Element ◽  
Cell Types ◽  
Intratumoral Heterogeneity ◽  
Gene Promoters ◽  
Cpg Island Methylator Phenotype ◽  
Reduced Representation

Abstract Genetic and epigenetic alterations contribute to the observed intratumoral heterogeneity in adult glioma. Current glioma classification, based on genotype (e.g., IDH1 mutations) and DNA methylation profiles (e.g., glioma CpG Island Methylator Phenotype), can provide clinically relevant tumor subgroups. However, traditional bulk sampling fails to adequately capture the full complement of epigenomic heterogeneity, and may mask deadly features present in less abundant glioma cells. To more precisely characterize the glioma epigenome, we separately profiled single-cell DNA methylation (Reduced Representation Bisulfite Sequencing, RRBS), single-cell RNA expression (10X genomics), and bulk whole genome sequencing in nine gliomas. The genomic regions profiled by scRRBS were primarily gene promoters, but adequate coverage was also reached for glioma-specific enhancer elements and binding sites of chromatin remodelers. Unsupervised clustering of single-cell DNA methylation data revealed intratumoral variability in epigenetic classification and these cell types were distinguished by regulatory element DNA methylation. We further integrated single-cell epigenetic, single-cell transcriptomic, and genomic features to better understand gene regulation and reconstruct each tumor’s lineage history. Together, our study aims to generate a glioma cellular hierarchy shaped by the epigenetic programs that drive tumor growth.

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