Reduced representation bisulfite sequencing data of mouse genome
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE76238 | GEO | 2016/02/26
SECONDARY ACCESSION(S): PRJNA306687
REPOSITORIES: GEO
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Project description:Reduced representation bisulfite sequencing (RRBS) has been proven a powerful method in DNA methylome profiling. Since the initial development of this method, the RRBS protocol has been modified in order to optimize it for genomic coverage, starting material, and library-construction throughput, which has resulted in new methods such as enhanced RRBS (ERRBS), double-enzyme RRBS (dRRBS), gel-free and multiplexed RRBS (mRRBS), and single-cell RRBS (scRRBS). However, each of these methods has failed to address PCR-derived duplication artifacts, which can bias the results of DNA methylation analyses. To overcome the aforementioned complication, we developed quantitative RRBS (Q-RRBS), a method in which unique molecular identifiers (UMIs) are used to eliminate PCR-induced duplication. By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.
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Project description:Differential hyper- and hypo-methylation regions in G0 versus G4/G5 CMP The goal of this study is to evaluate changes in CpG methylation profilings of telomere dysfunctional common myeloid progenitor cells (CMP) as compared to their wild type controls Genomic DNA was extracted from sorted CMP populations isolated from 3 pools of G0 or 2 pools of G5 mice using UltraPure Phenol:Chloroform:Isoamyl Alcohol according to manufacturer’s instructions (Life Technologies). 14,000 to 30,000 cells were available for each sample, resulting in a minimum of 45ng of DNA. Genome-wide DNA methylation profiling was performed by RRBS. Library preparation and sequencing were performed at the UT MD Anderson Cancer Center’s DNA Methylation Analysis Core and Sequencing and Microarray Facility, according to published protocols. RRBS sequencing data were aligned and methylation was called using Bismark v0.7.119. In brief, bisulphite-treated DNA was aligned to UCSC Genome Browser mm10 reference genome using Bowtie. In total 29-38 million reads were generated per sample with alignment rates around 63%. Next, MethylKit10 implemented with Fisher’s exact test was used to compare the cytosine methylation profiles of G0 and G5 CMP. Gene promoter regions were calculated based on RefSeq gene annotations with regions starting 1 kb upstream of the annotated transcription start site (TSS) and extending 500 base pairs downstream of TSS. Exons, introns, and CpG islands coordinates were collected from the UCSC Genome Browser mm10 version.
2015-04-09 | E-GEOD-62392 | biostudies-arrayexpress
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Florencia Pauli mailto:fpauli@hudsonalpha.org). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track is produced as part of the ENCODE project. The track reports the percentage of DNA molecules that exhibit cytosine methylation at specific CpG dinucleotides. 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 is assayed at more than 500,000 CpG dinucleotides in the genome using Reduced Representation Bisulfite Sequencing (RRBS). Genomic DNA is digested with the methyl-insensitive restriction enzyme MspI, small genomic DNA fragments are purified by gel electrophoresis, and then used to construct an Illumina sequencing library. The library fragments are treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymidine while leaving methylated cytosines intact. The sequenced fragments are aligned to a customized reference genome sequence and for each assayed CpG we report the number of sequencing reads covering that CpG and the percentage of those reads that are methylated. 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 CpG sites was assayed with a modified version of Reduced Representation Bisulfite Sequencing (RRBS; Meissner et al., 2008). RRBS was performed on cell lines grown by many ENCODE production groups. The production group that grew the cells and isolated genomic DNA is indicated in the "obtainedBy" field of the metadata. When a cell type was provided by more than one lab, the data for the cells from only one lab are displayed in the table above. However, the data for every cell type from every lab is available from the Downloads page. RRBS was carried out by the Myers production group at the HudsonAlpha Institute for Biotechnology. Isolation of genomic DNA Genomic DNA is isolated from biological replicates of 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 are determined using fluorescent DNA binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). Typically, 1 µg of DNA is used to make an RRBS library; however, we have also had success in making libraries with 200 ng genomic DNA from rare or precious samples. RRBS library construction and sequencing RRBS library construction starts with MspI digestion of genomic DNA , which cuts at every CCGG regardless of methylation status. Klenow exo- DNA Polymerase is then used to fill in the recessed end of the genomic DNA and add an adenosine as a 3prime overhang. Next, a methylated version of the Illumina paired-end adapters is ligated onto the DNA. Adapter ligated genomic DNA fragments between 105 and 185 basepairs are selected using agarose gel electrophoresis and Qiagen Qiaquick Gel Extraction Kit. The selected adapter-ligated fragments are 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 is amplified in a final PCR reaction which has been optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction uracils are copied as thymines resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. The sample is now ready for sequencing on the Illumina sequencing platform. These libraries were sequenced with an Illumina Genome Analyzer IIx according to the manufacturer's recommendations. Data analysis To analyze the sequence data, a reference genome is created that contains only the 36 base pairs adjacent to every MspI site and every C in those sequences is changed to T. A converted sequence read file is then created by changing each C in the original sequence reads to a T. The converted sequence reads are aligned to the converted reference genome, and only reads that map uniquely to the reference genome are kept. Once reads are aligned the percent methylation is calculated for each CpG using the original sequence reads. The percent methylation and number of reads is reported for each CpG.
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