scholarly journals "Same Difference": Comprehensive evaluation of three DNA methylation measurement platforms

2016 ◽  
Author(s):  
Thadeous J Kacmarczyk ◽  
Mame P. Fall ◽  
Xihui Zhang ◽  
Yuan Xin ◽  
Yushan Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Restriction Enzyme ◽  
Comprehensive Evaluation ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Starting Material ◽  
Reduced Representation ◽  
Targeted Capture ◽  
Areas Of Interest

ABSTRACTBackgroundDNA methylation in CpG context is fundamental to the epigenetic regulation of gene expression in high eukaryotes. Disorganization of methylation status is implicated in many diseases, cellular differentiation, imprinting, and other biological processes. Techniques that enrich for biologically relevant genomic regions with high CpG content are desired, since, depending on the size of an organism’s methylome, the depth of sequencing required to cover all CpGs can be prohibitively expensive. Currently, restriction enzyme based reduced representation bisulfite sequencing and its modified protocols are widely used to study methylation differences. Recently, Agilent Technologies and Roche NimbleGen have ventured to both reduce sequencing costs and capture CpGs of known biological relevance by marketing in-solution custom-capture hybridization platforms. We aimed to evaluate the similarities and differences of these three methods considering each targets approximately 10-13% of the human methylome.ResultsOverall, the regions covered per platform were as expected: targeted capture based methods covered >95% of their designed regions whereas the restriction enzyme-based method covered >70% of the expected fragments. While the total number of CpG loci shared by all methods was low, ~30% of any platform, the methylation levels of CpGs common across platforms were concordant. Annotation of CpG loci with genomic features revealed roughly the same proportions of feature annotations across the three platforms. Targeted capture methods encompass similar amounts of annotations with the restriction enzyme based method covering fewer promoters (~9%) and shores (~8%) and more unannotated loci (7-14%).ConclusionsAlthough all methods are largely consistent in terms of covered CpG loci and cover similar proportions of annotated CpG loci, the restriction based enrichment results in more unannotated regions and the commercially available capture methods result in less off-target regions. Quality of DNA is very important for restriction based enrichment and starting material can be low. Conversely, quality of the starting material is less important for capture methods, and at least twice the amount of starting material is required. Pricing is marginally less for restriction based enrichment, and number of samples to be prepared is not restricted to the number of samples a kit supports. The one advantage of capture libraries is the ability to custom design areas of interest. The choice of the technique should be decided by the number of samples, the quality and quantity of DNA available and the biological areas of interest since comparable data are obtained from all platforms.

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 Targeted Bisulfite Sequencing Reveals DNA Methylation Changes in Zinc Finger Family Genes Associated With KRAS Mutated Colorectal Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Weilin Pu ◽  
Fei Qian ◽  
Jing Liu ◽  
Keke Shao ◽  
Feng Xiao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Early Diagnosis ◽  
Zinc Finger ◽  
Comprehensive Evaluation ◽  
Methylation Status ◽  
Area Under The Curve ◽  
Validation Dataset ◽  
Kras Mutations ◽  
Diagnostic Capability

Background: Colorectal cancer (CRC) is a leading cause of cancer death, and early diagnosis of CRC could significantly reduce its mortality rate. Previous studies suggest that the DNA methylation status of zinc finger genes (ZFGs) could be of potential in CRC early diagnosis. However, the comprehensive evaluation of ZFGs in CRC is still lacking.Methods: We first collected 1,426 public samples on genome-wide DNA methylation, including 1,104 cases of CRC tumors, 54 adenomas, and 268 para-tumors. Next, the most differentially methylated ZFGs were identified and validated in two replication cohorts comprising 218 CRC patients. Finally, we compared the prediction capabilities between the ZFGs and the SEPT9 in all CRC patients and the KRAS + and KRAS- subgroup.Results: Five candidate ZFGs were selected: ESR1, ZNF132, ZNF229, ZNF542, and ZNF677. In particular, ESR1 [area under the curve (AUC) = 0.91] and ZNF132 (AUC = 0.93) showed equivalent or better diagnostic capability for CRC than SEPT9 (AUC = 0.91) in the validation dataset, suggesting that these two ZFGs might be of potential for CRC diagnosis in the future. Furthermore, we performed subgroup analysis and found a significantly higher diagnostic capability in KRAS + (AUC ranged from 0.97 to 1) than that in KRAS- patients (AUC ranged from 0.74 to 0.86) for all these five ZFGs, suggesting that these ZFGs could be ideal diagnostic markers for KRAS mutated CRC patients.Conclusion: The methylation profiles of the candidate ZFGs could be potential biomarkers for the early diagnosis of CRC, especially for patients carrying KRAS mutations.

scholarly journals MINI REVIEW: Statistical methods for detecting differentially methylated loci and regions

2014 ◽  
Author(s):  
Mark D Robinson ◽  
Abdullah Kahraman ◽  
Charity W Law ◽  
Helen Lindsay ◽  
Malgorzata Nowicka ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Statistical Methods ◽  
Empirical Bayes ◽  
Large Scale ◽  
Rapid Development ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Data Types ◽  
Cpg Dinucleotides ◽  
Reversible Addition

DNA methylation, and specifically the reversible addition of methyl groups at CpG dinucleotides genome-wide, represents an important layer that is associated with the regulation of gene expression. In particular, aberrations in the methylation status have been noted across a diverse set of pathological states, including cancer. With the rapid development and uptake of large scale sequencing of short DNA fragments, there has been an explosion of data analytic methods for processing and discovering changes in DNA methylation across diverse data types. In this mini-review, we aim to condense many of the salient challenges, such as experimental design, statistical methods for differential methylation detection and critical considerations such as cell type composition and the potential confounding that can arise from batch effects, into a compact and accessible format. Our main interests, from a statistical perspective, include the practical use of empirical Bayes or hierarchical models, which have been shown to be immensely powerful and flexible in genomics and the procedures by which control of false discoveries are made. Of course, there are many critical platform-specific data preprocessing aspects that we do not discuss here. In addition, we do not make formal performance comparisons of the methods, but rather describe the commonly used statistical models and many of the pertinent issues; we make some recommendations for further study.

305 SPERM CHROMATIN STRUCTURE AND DNA METHYLATION BULLS

2006 ◽  
Vol 18 (2) ◽  
pp. 260
Author(s):  
K. Hengstberger ◽  
N. Phutikanit ◽  
O. Berking ◽  
M. D'Occhio ◽  
D. Sester ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Conformational Changes ◽  
Chromatin Structure ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Bos Indicus ◽  
Methylation Pattern ◽  
Embryonic Mortality ◽  
Sperm Chromatin ◽  
Detergent Solution

Mammalian spermatogenesis is a complex process that involves genome-wide deprogramming and reprogramming of DNA methylation at different stages in order to produce sperm that are capable of fertilization and also TO support ongoing embryo development (Webster et al. 2005 PNAS 102, 4068-4073). Embryonic mortality occurs when there is inappropriate expression of developmentally regulated genes of both maternal and paternal origin. DNA methylation is associated with epigenetic regulation of gene expression and it was recently shown that aberrant DNA methylation causes a change in sperm chromatin structure (Webster et al. 2005). Studies, primarily in man, have indicated that sperm chromatin instability, as determined by the sperm chromatin structure assay (SCSA), does not influence fertilization but is linked with early pregnancy failure (Evenson and Jost 2000 Methods Cell Sci. 22, 169-189). The aim of the present study was to investigate the relationship between chromatin structure and DNA methylation in bull sperm. Chromatin structure was determined using the SCSA that involves the exposure of sperm to a low pH (1.2) detergent solution followed by the addition of acridine orange (AO). When exposed to a 488 nm laser, AO fluoresces green when bound to native (double-stranded) DNA and red when bound to denatured (single-stranded) DNA. The SCSA yields a value for the DNA fragmentation index (DFI), and in men a DFI > 27-30% is associated with early embryonic mortality (Larson-Cook et al. 2003 Fertil. Steril. 80, 895-902). Semen was obtained by electroejaculation from Zebu (Bos indicus) bulls (n = 12) in a subtropical environment; a proportion of bulls had a DFI < 15% (n = 4) and the remainder a DFI > 27% (n = 8). The DNA methylation pattern, as determined by the amplified methylation polymorphism (AMP) protocol (Webster et al. 2005), appeared to differ between sperm with a DFI < 15% and sperm with a DFI > 27% when assessed by principle coordinate analysis. Also, sperm with a DFI < 15% had a more consistent methylation pattern compared with apparent differences in methylation among sperm with a DFI > 27%. These preliminary findings could be interpreted to suggest that methylation status can contribute to chromatin structure in sperm of mature bulls. It remains to be determined whether the environment can influence sperm chromatin status in bulls by altering DNA methylation which then impacts on the conformational changes in DNA that occur during spermiogenesis. This work was supported, in part, by Meat and Livestock Australia.

scholarly journals Genome Wide MeDIP-Seq Profiling of Wild and Cultivated Olives Trees Suggests DNA Methylation Fingerprint on the Sensory Quality of Olive Oil

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1405
Author(s):  
Oussama Badad ◽  
Naoufal Lakhssassi ◽  
Nabil Zaid ◽  
Abdelhalim El Baze ◽  
Younes Zaid ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Phenotypic Plasticity ◽  
Secondary Metabolites ◽  
Olive Oil ◽  
Sensory Quality ◽  
Methylation Status ◽  
Genome Wide ◽  
Pharmaceutical Properties ◽  
Methylation Profiling

Secondary metabolites are particularly important to humans due to their pharmaceutical properties. Moreover, secondary metabolites are key compounds in climate change adaptation in long-living trees. Recently, it has been described that the domestication of Olea subspecies had no major selection signature on coding variants and was mainly related to changes in gene expression. In addition, the phenotypic plasticity in Olea subspecies was linked to the activation of transposable elements in the genes neighboring. Here, we investigated the imprint of DNA methylation in the unassigned fraction of the phenotypic plasticity of the Olea subspecies, using methylated DNA immuno-precipitation sequencing (MeDIP-seq) for a high-resolution genome-wide DNA methylation profiling of leaves and fruits during fruit development in wild and cultivated olives from Turkey. Notably, the methylation profiling showed a differential DNA methylation in secondary metabolism responsible for the sensory quality of olive oil. Here, we highlight for the first time the imprint of DNA methylation in modulating the activity of the Linoleate 9S lipoxygenase in the biosynthesis of volatile aromatic compounds. Unprecedently, the current study reveals the methylation status of the olive genome during fruit ripening.

scholarly journals The role of DNA methylation in ageing and cancer

2018 ◽  
Vol 77 (4) ◽  
pp. 412-422 ◽  
Author(s):  
A. E. Morgan ◽  
T. J. Davies ◽  
M. T. Mc Auley
Keyword(s):  
Dna Methylation ◽  
Gene Silencing ◽  
Methylation Status ◽  
Electrochemical Techniques ◽  
Regulation Of Gene Expression ◽  
Homeobox Gene ◽  
Gene Promoter ◽  
Disease States ◽  
Potential Biomarker ◽  
Aberrant Dna Methylation

The aim of the present review paper is to survey the literature related to DNA methylation, and its association with cancer and ageing. The review will outline the key factors, including diet, which modulate DNA methylation. Our rationale for conducting this review is that ageing and diseases, including cancer, are often accompanied by aberrant DNA methylation, a key epigenetic process, which is crucial to the regulation of gene expression. Significantly, it has been observed that with age and certain disease states, DNA methylation status can become disrupted. For instance, a broad array of cancers are associated with promoter-specific hypermethylation and concomitant gene silencing. This review highlights that hypermethylation, and gene silencing, of the EN1 gene promoter, a crucial homeobox gene, has been detected in various forms of cancer. This has led to this region being proposed as a potential biomarker for diseases such as cancer. We conclude the review by describing a recently developed novel electrochemical method that can be used to quantify the level of methylation within the EN1 promoter and emphasise the growing trend in the use of electrochemical techniques for the detection of aberrant DNA methylation.

Abstract 40: Transcriptome and DNA Methylation Changes in Patients with Subarachnoid Hemorrhage Undergoing Remote Ischemic Preconditioning

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Elina Nikkola ◽  
Arthur Ko ◽  
Mark J Connolly ◽  
Yinn Cher Ooi ◽  
Päivi Pajukanta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Profiles ◽  
Methylation Status ◽  
Gene Expression Profiles ◽  
Reduced Representation ◽  
Ischemic Conditioning ◽  
Genome Wide ◽  
Reduced Representation Bisulfite Sequencing ◽  
Before And After

Background: Remote ischemic conditioning (RIC) is a phenomenon by which brief periods of sublethal ischemia in one tissue confers protection from ischemia to distant tissues. We hypothesize that RIC triggers a cascade of integrated gene expression and methylation changes, leading to neuroprotection in subarachnoidal hemorrhage (SAH) patients. Our goal was to identify and compare changes in DNA methylation and gene expression profiles before and after RIC. Methods: Patients enrolled in a clinical trial of RIC after SAH, receiving RIC by limb cuff transient ischemia sessions. Fourteen SAH patients (64% female, mean age 51) underwent 3-4 RIC sessions and gave a blood sample before and after RIC, seven days apart. The transcriptome analysis of whole blood was performed using paired-end, 100-bp RNA-sequencing. We employed STAR and HTSeq to align and count reads; EdgeR to normalize the counts and detect differential expression (DE); and David to search for functional categories of the DE genes. Genome-wide DNA methylation profiles were assessed using reduced representation bisulfite sequencing (RRBS); Bismark with Bowtie to align the RRBS data, and the differential methylation analysis package (DMAP) to call the methylation status of CpG sites. Bedtools was used to overlap the DE genes with differentially methylated regions. Results: Of the 12,411 genes passing QC, 168 genes were differentially expressed after RIC (FDR<0.05). These genes were enriched for pathways involving mitosis and nuclear division (P50% after RIC in at least one individual. Of the 8,069 sites, 723 were differentially methylated (Bonferroni P<0.05). Our overlap analysis showed that 88 of the significantly altered methylation sites resided in 39 DE genes, including CEACAM8 and CRISP3, both implicated previously for stroke. Conclusions: Our data suggest that RIC alters expression of a specific set of genes involved in stroke via changes in regional DNA methylation. Further studies are warranted to replicate these pilot results.

scholarly journals Genes of the month: H3.3 histone genes: H3F3A and H3F3B

2021 ◽  
pp. jclinpath-2021-207857
Author(s):  
Vishnu Chandra Kumar ◽  
Rekha Pai
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Developmental Disorders ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Histone Genes ◽  
Histone Tails ◽  
Post Translational Modifications ◽  
Altered Gene ◽  
Methylation Patterns

Histones constitute the chief protein component of DNA. They help to maintain chromatin structure and regulate gene expression. The long double-stranded DNA molecule winds around histone octamers to form nucleosomes which serve the purpose of compacting DNA within the confines of the nuclear membrane. There are five major types of histones, namely H1/H5, H2, H3 and H4. H3.3 is a subtype of H3 histone and can be encoded either by the H3F3A or H3F3B genes independently. Amino acids such as lysine and arginine found in the histone tails are sites of post-translational modifications (PTMs) such as methylation and acetylation. These PTMs in histones are involved in the regulation of gene expression by chromatin remodelling and by controlling DNA methylation patterns. Mutations in histone genes can affect sites of PTMs causing changes in local and global DNA methylation status. These effects are directly linked to neoplastic transformation by altered gene expression. Recurrent H3.3 histone mutations are increasingly identified in several malignancies and developmental disorders. The following review attempts to shed light on the diseases associated with H3.3 histone mutations.

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