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96 mRRBS libraries: Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA

ABSTRACT: Sequencing based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage as the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies including cohorts of cancer samples.

ORGANISM(S): Homo sapiens

PROVIDER: GSE40429 | GEO | 2012/08/29

SECONDARY ACCESSION(S): PRJNA173930

REPOSITORIES: GEO

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96 mRRBS libraries: Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA

Project description:Sequencing based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage as the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies including cohorts of cancer samples. Libraries of 96 human samples

2012-08-29 | E-GEOD-40429 | biostudies-arrayexpress

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Project description:This study is aimed to explore whether C1qbp deficiency affects the DNA methylation status during the differentiation stage of CD8+ T cells. Transferred WT and C1qbp KO P14 cells sorted from the spleen of donor mice on day 5 post-infection with LCMV Armstrong were collected. Genomic DNA was isolated for mRRBS. mRRBS analysis provided evidence of DNA methylation changes between WT and C1qbp KO CD8+ T cells. Our study have shown that, on day 5 post-infection with LCMV Armstrong, C1qbp KO CD8+ T cells have increased level of Global DNA methylation. Moreover, C1qbp KO CD8+ T cells show hegher DNA methylation at some effector cells-signature genes.

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2018-03-06 | GSE111000 | GEO

Quantitative reduced representation bisulfite sequencing with unique molecular identifiers of single cell

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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Differential DNA Methylation Analysis without a Reference Genome

Project description:Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for studying DNA methylation in non-model organisms, we developed an integrated approach for studying DNA methylation differences without a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, zebrafish, carp, and sea bass). Bioinformatically, we developed the RefFreeDMA software (http://RefFreeDMA.computational-epigenetics.org) to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions. These regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

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Project description:We developed a high-throughput sequencing protocol that efficiently captures small RNAs from low input samples while minimizing inherent biases associated with library production. The protocol was builds on previously published method that adds a barcode to samples at the earliest step in preparation such that all downstream manipulations can be performed on a pool of many samples thereby greatly reducing reagent usage and workload. We optimized adapter concentrations and showed modified adapters greatly increases the efficiency of small RNA capture. This work also demonstrates that unique molecular identifiers incorporated into the adapters can be used to bioinformatically remove PCR duplication.

2019-01-31 | GSE109162 | GEO

Homo sapiens

Project description:96 mRRBS libraries: Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA

| PRJNA173930 | ENA

Mus musculus

Project description:Pregnancy-associated alterations in DNA methylation patterns of mammary epithelial stem cells [mRRBS]

| PRJNA235306 | ENA

Genome-wide DNA methylation change due to lncRNA H19 knockdown in myotubes

Project description:We used Methyl-MiniSeq platform from Zymo Research company to identify genome-wide methylation changes affected by lncRNA H19 knockdown in myotubes. Following H19 knockdown, we observed extensive genome-wide mthylation pattern changes relative to siCon cells, with some genes showing incresed methylation, others showing decreased methylation, and a third group with no significant change. Myotubes differentiated from mouse C3H myoblasts were transfected with either control siRNA or siH19, 48h later, cellular genomic DNA was extracted and subjected to genome-scale DNA methylation mapping using the platform of an improved version of Reduced Representation Bisulfite Sequencing (RRBS).

2015-11-09 | E-GEOD-73013 | biostudies-arrayexpress

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