Methylated DNA is over-represented in whole-genome bisulfite sequencing data
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE58217 | GEO | 2014/09/11
SECONDARY ACCESSION(S): PRJNA251597
REPOSITORIES: GEO
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Project description:The development of whole-genome bisulfite sequencing (WGBS) has led to a number of exciting discoveries about how genomes utilize DNA methylation and has led to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently excelled to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of methylated DNA from WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. MethylC-Seq of Arabidopsis thaliana
2014-09-11 | E-GEOD-58217 | biostudies-arrayexpress
Project description:Cannabis is commonly used amongst reproductive age males. Our group and others have shown that chronic delta-9-tetrahydrocannabinol (THC) use adversely impacts male fertility, but less is known about the potential reversibility of these changes. Our study’s objective was to determine if THC discontinuation mitigates THC-associated changes in male reproductive health using a rhesus macaque (RM) model of daily THC edible consumption over a 280-day period (4 spermatogenic cycles). Testicular volume, serum male hormones, semen parameters, sperm DNA fragmentation, seminal fluid proteomics, and whole genome bisulfite sequencing (WGBS) of sperm DNA were assessed at pre-THC, moderate-THC, and heavy-THC dosing, and at 70 and 140 days after THC discontinuation. Chronic THC use resulted in significant testicular atrophy, increased gonadotropins, decreased serum sex steroids, and increased DNA fragmentation. THC discontinuation led to partial recovery of testicular volume, sex steroids and sperm DNA integrity. Seminal fluid proteome analysis revealed differential expression of proteins enriched for processes related to cellular secretion, immune response, and fibrinolysis. WGBS identified significant differential methylation in heavy-THC versus pre-THC sperm, with partial restoration of methylation after discontinuation of THC. Genes associated with differentially methylated regions (DMRs) were enriched for pathways involved in nervous system development and function. This is the first study demonstrating that chronic THC use in RMs adversely impacts male reproductive health, methylation of genes involved in development, and expression of proteins important for male fertility. THC discontinuation improves impacts to male fertility, including partial restoration of THC-associated sperm DMRs in genes important for development.
2023-05-18 | PXD039802 | Pride
Project description:We carry out whole-genome bisulfite sequencing (WGBS) of rat placental DNA to investigate epigenome-wide alterations in a reduced uterine perfusion pressure (RUPP) PE model. The Hidden Markov Model (HMM) was applied to identify functional epigenetic structures including partially methylated domains (PMDs), low methylated regions (LMRs) and unmethylated regions (UMRs).
2023-08-12 | GSE211194 | GEO
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track was produced as part of the ENCODE project. It reports the percentage of DNA molecules that exhibit cytosine methylation. In general, DNA methylation within a gene's promoter is associated with gene silencing, and DNA methylation within the exons and introns of a gene is associated with gene expression. Proper regulation of DNA methylation is essential during development and aberrant DNA methylation is a hallmark of cancer. DNA methylation status was assayed with Whole Genome Bisulfite Sequencing (WGBS). Genomic DNA was sheared by sonication, end-repaired and then ligated to methylated sequencing adapters. The library fragments were treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymine while leaving methylated cytosines intact. The sequenced fragments were aligned to a bisulfite-converted reference genome. For each assayed cytosine, the number of sequencing reads covering that C and the percentage of those reads that were methylated were reported. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf DNA methylation at cytosines across the genome was assayed with Whole Genome Bisulfite Sequencing (WGBS). WGBS was performed on cell lines grown by ENCODE production groups. WGBS was carried out by the Myers production group at the HudsonAlpha Institute for Biotechnology. Isolation of Genomic DNA: Genomic DNA was isolated from each cell line using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations for each genomic DNA preparation were determined using fluorescent DNA-binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). Typically, 2 µg of genomic DNA is used to make WGBS libraries. WGBS Library Construction and Sequencing: WGBS library construction started with sonication of genomic DNA on a Covaris S2 instrument. Sheared ends were then repaired and blunted with DNA polymerase I, T4 DNA polymerase and T4 polynucleotide kinase in the presence of dATP, dGTP and dTTP. After end repair, Klenow exo- DNA Polymerase was used to add an adenosine as a 3' overhang. Next, a methylated version of the Illumina paired-end adapters was ligated onto the DNA. Adapter-ligated 400 bp genomic DNA fragments were selected using a 2% agarose SizeSelect E-gel. The selected adapter-ligated fragments were treated with sodium bisulfite using the Zymo Research EZ DNA Methylation Gold Kit, which converts unmethylated cytosines to uracils and leaves methylated cytosines unchanged. Bisulfite-treated DNA was amplified in a final PCR reaction which was optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction, uracils were copied as thymines, resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. These libraries were then sequenced with an Illumina HiSeq 2000 according to the manufacturer's recommendations as paired-end 50 bp reads. Libraries were sequenced to a depth of 600 million aligned reads. Data Analysis: To analyze the sequence data, Bismark (Krueger and Andrews, 2011) was used to align sequences reads. Generally, each read went through a conversion of Cs to Ts and was then aligned to fully converted plus and minus strands of the hg19 build of the human genome. A few custom refinements were made to the Bismark program. Since these libraries were made in a directional orientation with the first read always being C-poor, we skipped unnecessary alignments to impossible orientations. We also implemented a more stringent uniqueness filter, only allowing reads that have one acceptable alignment (based on default Bowtie parameters) across both strands. Once reads were aligned, the percent methylation was calculated for each cytosine using the original sequence reads. The percent methylation and number of reads is reported for each CpG in the wgEncodeHaibMethylWgbsXXXXCpg.bigBed file and for each non CpG cytosine in the wgEncodeHaibMethylWgbsXXXXNoncpg.bigBed file.
2012-09-12 | E-GEOD-40832 | biostudies-arrayexpress
Project description:Examination of two DNA methylomes at the most extreme points of their lives to see differences that might contribute to explain the aged phenotype We have performed Whole Genome Bisulfite Sequencing (WGBS) and methylation array technology of newborn and a centenarian samples to address the Epigenetic drifts in Human aging, which might be an alternative pathway to explain the age-associated alterations. In addition, we used SNP array platforms to exclude an influence of copy number changes with age and SNPs on the identification of differentially methylated regions. This Series represents the methylation array data. The WGBS data are available in GSE31263.
2012-07-10 | E-GEOD-31438 | biostudies-arrayexpress
Project description:Whole genome bisulfite sequencing (WGBS) was used to assess the effect of M. bovis infection on the bAM DNA methylome (5-methylcytosine). The methylomes of bAM infected with M. bovis (n = 8) were compared to those of non-infected control bAM (n = 8) at 24 hours post-infection (hpi). No differences in DNA methylation (CpG or non-CpG) were observed between control and infected bAM. Analysis of DNA methylation at proximal promoter regions uncovered >250 genes harbouring intermediately methylated (IM) promoters (average methylation = 33–66%). Gene ontology (GO) analysis, focusing on genes with low, intermediate or highly methylated promoters, revealed that genes with IM promoters were enriched for immune-related GO categories; this enrichment was not observed for genes in the high or low methylation groups. Targeted analysis of two non-imprinted genes in the IM category, C1QB and IL2RA, confirmed the WGBS observation. This study is the first in cattle to examine genome-wide DNA methylation at single nucleotide resolution in an important bovine cellular host-pathogen interaction model and provides evidence for intermediate promoter methylation in bAM.
2019-02-13 | GSE110412 | GEO
Project description:Whole-Genome Bisulfite Sequencing of HCHC mutants To extend our understanding of the role of the HCHC complex in Neurospora, we carried out whole-genome bisulfite sequencing (WGBS) of cdp-2, chap, and hda-1 mutants. Consistent with prior analyses, the WGBS revealed both hypomethylated and hypermethylated regions in the three HCHC mutants while the ; sequences with a lower Combined RIP Index (CRI) tend to show reduced methylation in the mutants, while sequences with higher CRI scores show increased methylation. Analysis of the WGBS data also demonstrated that shorter methylated regions, regardless of their degree of methylation in wild type, commonly have reduced methylation in the HCHC mutants, while longer methylated sequences tend to have elevated methylation. In addition, the borders of normally methylated regions typically lose methylation and show a contraction of boundary methylation in the HCHC mutants. Sequences near telomeres that are normally methylated were found to lose methylation in the mutants, while most of the increased DNA methylation of the three HCHC mutants is found at centromeres. CHAP in vitro DNA-affinity purification HT-seq and CHAP DamID-Seq To test whether the CHAP AT-hook motifs bind AT-rich RIP’d DNA, we performed in vitro DNA-affinity purification with the recombinant CHAP-N-terminus (1-274 residues) containing the two AT-hook motifs and analyzed the purified DNA with high throughput sequencing (HT-Seq). To complement this approach, we also assessed binding of CHAP in vivo with DamID-seq using the CHAP-Dam strain. Together, these techniques gave us a detailed genomic view of the specific localization and binding of CHAP to AT-rich RIP’d DNA, which is nearly coincident with methylated DNA regions.
2017-01-01 | GSE81129 | GEO
Project description:Purpose: To investigate the effect of Tet1 depletion on global DNA methylation, we performed whole-genome bisulfite sequencing (WGBS). Methods: Starting with as little as 1400-5251 manually micro-dissected PGCs, we used an ultra-low input method, Tn5mC-seq. Results: We generated 945 million reads for Tet1Gt/Gt PGCs and 302 million reads for wild-type PGCs. We obtained 14-16 million CpG sites per genotype at 1.76-2.66x genome coverage, which enables a comprehensive view of genome-wide DNA methylation patterns in E13.5 PGCs. PGCs are almost completely unmethylated genome-wide. Loss of Tet1 led to a subtle increase of methylation level in various genomic elements including promoters, exons, introns and repetitive elements in Tet1Gt/Gt PGCs (p<0.01). Local analysis identified 4,337 differentially methylated regions (DMRs) between Tet1-/- PGCs and wild-type cells. These DMRs are associated with 5,261 genes, among which 271 genes also exhibited differential gene expression and enriched for the cell cycle pathway (FDR=0.02). Conclusions: This result revealed that demethylation of certain set of cell cycle genes is largely abolished in the Tet1-/- PGCs. Genome-wide methylation profiles of primordial germ cells derived from the wild type (WT) and Tet1-null female embryos at E13.5 were generated by whole genome bisulfite sequencing using Illumina Hiseq.
2012-11-20 | E-GEOD-41912 | biostudies-arrayexpress
Project description:Whole-genome bisulfite sequencing (WGBS) is currently the gold standard for DNA methylation (5-methylcytosine, 5mC) profiling, however the destructive nature of sodium bisulfite results in DNA fragmentation and subsequent biases in sequencing data. Such issues have led to the development of bisulfite-free methods for 5mC detection. Nanopore sequencing is a long read non-destructive approach that directly analyzes DNA and RNA fragments in real time. Recently, computational tools have been developed that enable base-resolution detection of 5mC from Oxford Nanopore sequencing data. In this chapter we provide a detailed protocol for preparation, sequencing, read assembly and analysis of genome-wide 5mC using Nanopore sequencing technologies.
2021-12-03 | GSE179673 | GEO
Project description:Whole Genome Bisulfite Sequencing (WGBS) is the current standard for DNA methylation profiling; however, WGBS is costly as it requires sequencing coverage over the entire genome. Here we introduce Anchor-Based Bisulfite Sequencing (ABBS). We show that ABBS captures accurate DNA methylation information in Escherichia coli and mammalian cells, while requiring approximately 20 times fewer sequencing reads than WGBS. The ABBS protocol is simple and can be performed in a single day.
2022-05-23 | GSE180796 | GEO