Evaluation of the whole genome amplification of bisulfite modified DNA
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE39565 | GEO | 2015/07/22
SECONDARY ACCESSION(S): PRJNA171142
REPOSITORIES: GEO
Similar Datasets
Project description:We compared genom-wide DNA methylation profiles between whole genome amplified bisufite-modified DNA and non-amplified DNA. 3 samples were bisulfite-converted, non-amplified DNA (technical triplicate). 6 samples were amplified with EpiTect® Whole Bisulfitome from 10ng or 50ng of bisulfite-converted DNA. All samples were hybridized to the Illumina Infinium HumanMethylation450.
2015-07-22 | E-GEOD-39565 | biostudies-arrayexpress
Project description:UnlabelledBackgroundStudying DNA methylation profiles in detail should be the first step in epigenetic research. Although sodium bisulfite modification of genomic DNA is the gold standard method for DNA methylation analysis, this method results in the loss of the majority of the DNA material. Whole genome amplification (WGA) of bisulfite-modified DNA is expected to provide a rich source of materials, but its validity has not been thoroughly evaluated. In this study, we evaluated the extent of biased amplification in the WGA of bisulfite-modified DNA and the reproducibility of independent WGA reactions. We performed the multiple displacement amplification-based WGA separately three times. Each experiment included two reactions using 10 or 50 ng of bisulfite-modified DNA as template. DNA methylation levels were compared between WGA products and original bisulfite-modified DNA at about 450,000 CpG sites.ResultsUsing a sufficient amount of bisulfite-modified DNA for WGA was critical for downstream application. The considerable deviations from original bisulfite-modified DNA were found in the middle range of DNA methylation levels. Distribution of hyper- and hypomethylation were equal, which suggested that the deviation at each CpG site occurred randomly. Averaging the data from independently amplified WGA products dramatically improved the overall quality.ConclusionsWGA of bisulfite-modified DNA could be a valuable tool for epigenetic research, but careful experimental design and data interpretation are required.
| S-EPMC3536718 | biostudies-literature
Project description:DNA methylation plays a key role in epigenetic regulation of eukaryotic genomes. Hence the genome-wide distribution of 5-methylcytosine, or the methylome, has been attracting intense attention. In recent years, whole-genome bisulfite sequencing (WGBS) has enabled methylome analysis at single-base resolution. However, WGBS typically requires microgram quantities of DNA as well as global PCR amplification, thereby precluding its application to samples of limited amounts. This is presumably because bisulfite treatment of adaptor-tagged templates, which is inherent to current WGBS methods, leads to substantial DNA fragmentation. To circumvent the bisulfite-induced loss of intact sequencing templates, we conceived an alternative method termed Post-Bisulfite Adaptor Tagging (PBAT) wherein bisulfite treatment precedes adaptor tagging by two rounds of random primer extension. The PBAT method can generate a substantial number of unamplified reads from as little as subnanogram quantities of DNA. It requires only 100 ng of DNA for amplification-free WGBS of mammalian genomes. Thus, the PBAT method will enable various novel applications that would not otherwise be possible, thereby contributing to the rapidly growing field of epigenomics.
| S-EPMC3458524 | biostudies-literature
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
2012-09-12 | GSE40832 | GEO
Project description:Abstract: BACKGROUND: T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. RESULTS: Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. CONCLUSION: T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. This SuperSeries is composed of the SubSeries listed below.
2006-03-07 | GSE4349 | GEO
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.
2014-09-11 | GSE58217 | GEO
Project description:Since DNMT3A is a de novo DNA methyltransferase, transcriptional differences in WT versus Dnmt3a-/- HSCs upon infection suggest a possible role for epigenetic regulation in the HSC transcriptional response to inflammation. Therefore, we wanted to investigate if differences in stress and presence of Dnmt3a affect the methylome overall, and specific IFNg responses genes. 100000 LT-HSCs (LK CD150+ CD48- were sorted into lysis buffer from the pools of naive or 1-month (M. avium) infected WT/ Dnmt3-/-_x0001_ mice (n = 7-8 per group). Around 300 ng of DNA per group was extracted with AllPrep DNA/RNA Mini Kit. NEBNext Ultra II DNA library prep kit was used for library preparation. Bisulfite conversion step was added after ‘‘methylated’’ adaptor ligation and USER excision. Then, methylated adaptor ligated DNA fragment was treated with bisulfite conversion using EZ DNA methylationlightning kit. These modified DNA fragments were then amplified with index primers with Kapa HiFi urasil+ specialized polymerase for bisulfite converted DNA. For WGBS library sequencing, 150-high output kit from illumine was used. Samples (400 million reads/ sample (15-20X coverage)) were run in NovaSeq S1 FC (2, lanes 1,600 Million reads). Global methylation of Dnmt3a-/- cells was more divergent upon infection compared to WT. There were vastly more hyper- and hypomethylated regions in infected versus naive Dnmt3a-/- HSCs compared to WT. Among the altered regions, both WT and Dnmt3a-/- HSCs showed slightly more hypomethylated than hypermethylated regions, although changes in both directions were present. In the WT background, regions of hyper- and hypomethylation were mostly restricted to CpG islands. By contrast, highly significant differences in hypomethylation were found in the Dnmt3a-/- HSCs in CpG islands, shores, enhancers, and promoters DNMT3A is highly active in HSCs during infection and that the loss of DNMT3A function results in significant global changes in methylation, including both hyper- and hypomethylation, in genome regions that likely affect gene transcription. we examined the methylation of Batf2, Jun, and Fos. All 3 of these prodifferentiation genes showed increased methylation in the promoter region in the Dnmt3a-/- background as compared to the WT background. Hypermethylation of these promoter regions is consistent with their transcriptional repression.
2022-02-16 | GSE196896 | GEO
Project description:Abstract: Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalin-fixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. This SuperSeries is composed of the SubSeries listed below.
2006-03-15 | GSE4467 | GEO