Evaluation of methyl-binding domain based enrichment approaches revisited
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE64144 | GEO | 2014/12/20
SECONDARY ACCESSION(S): PRJNA270249
REPOSITORIES: GEO
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Project description:A comparison of methylMiner and MethylCap, two methylation enrichment kits The main analysis include two individuals (mice) in technical duplicates from various lab/technical conditions. The results are compared with previous studies of two human samples from a number of lab/technical conditions (human data available at dbGaP: record number phs000608.v1.p1). In addition a DNA from a third mouse is used for investigations of specific alterations of the protocol.
2014-12-20 | E-GEOD-64144 | biostudies-arrayexpress
Project description:Purpose: to compare different Methyl Binding Domain (MBD) based kits for DNA-methylation sequencing using Reduced Representation Bisulfite Sequencing (RRBS) data for validation, and to determine whether data quality can also be derived from inherent sequence data characteristics MBD-seq using 5 different kits (MethylCap, MethylCollector, MethylCollector Ultra, MethylMiner, MethylMagnet) was applied on 3 commonly used cell lines (DU145, HCT15, PC3), for which also RRBS data were generated.
2013-01-23 | E-GEOD-42790 | biostudies-arrayexpress
Project description:Methyl-binding domain (MBD) enrichment followed by deep sequencing (MBD-seq), is a robust and cost efficient approach for methylome-wide association studies (MWAS). MBD-seq has been demonstrated to be capable of identifying differentially methylated regions, detecting previously reported robust associations and producing findings that replicate with other technologies such as targeted pyrosequencing of bisulfite converted DNA. There are several kits commercially available that can be used for MBD enrichment. Our previous work has involved MethylMiner (Life Technologies, Foster City, CA, USA) that we chose after careful investigation of its properties. However, in a recent evaluation of five commercially available MBD-enrichment kits the performance of the MethylMiner was deemed poor. Given our positive experience with MethylMiner, we were surprised by this report. In an attempt to reproduce these findings we here have performed a direct comparison of MethylMiner with MethylCap (Diagenode Inc, Denville, NJ, USA), the best performing kit in that study. We find that both MethylMiner and MethylCap are two well performing MBD-enrichment kits. However, MethylMiner shows somewhat better enrichment efficiency and lower levels of background "noise". In addition, for the purpose of MWAS where we want to investigate the majority of CpGs, we find MethylMiner to be superior as it allows tailoring the enrichment to the regions where most CpGs are located. Using targeted bisulfite sequencing we confirmed that sites where methylation was detected by either MethylMiner or by MethylCap indeed were methylated.
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Project description:Purpose: to compare different Methyl Binding Domain (MBD) based kits for DNA-methylation sequencing using Reduced Representation Bisulfite Sequencing (RRBS) data for validation, and to determine whether data quality can also be derived from inherent sequence data characteristics
2013-01-23 | GSE42790 | GEO
Project description:DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD) based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1) to identify a best performing kit for MBD-based enrichment using independent validation data, and 2) to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3). Reduced representation bisulfite sequencing data (all three cell lines) and publicly available Illumina Infinium BeadChip data (DU145 and PC3) were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode's MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications.
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Project description:The DNA methylome of 45 primary neuroblastoma tumors is profiled by enrichment with a methyl-CpG-binding domain (MBD) and massively parallel sequencing DNA of 45 primary tumors is sheared (fragments of ± 200 bp), followed by MBD-based (MethylCap kit of Diagenode) enrichement, library preparation and multiplexing. Both input DNA and captured DNA were sequenced paired-end on Illumina Hiseq2000
2015-11-19 | E-GEOD-69243 | biostudies-arrayexpress
Project description:The DNA methylome of 15 primary stage 4S neuroblastoma tumors is profiled by enrichment with a methyl-CpG-binding domain (MBD) and massively parallel sequencing DNA of 15 primary stage 4S tumors is sheared (fragments of ± 200 bp), followed by MBD-based (MethylCap kit of Diagenode) enrichement, library preparation and multiplexing. Both input DNA and captured DNA were sequenced paired-end on Illumina Hiseq2000
2015-12-15 | E-GEOD-69268 | biostudies-arrayexpress
Project description:Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using MBD-enrichment are restricted to large (M-bM-^IM-%1 M-BM-5g) DNA samples, limiting the analysis of small tissue samples. Here we present a technique which enables characterization of genome wide tissue-specific methylation patterns from nanogram quantities of DNA. We have developed a methodology utilizing MBD2b/MBD3L1-enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized NimbleGen 2.1M mouse tiling arrays (Feinberg_MM8_Me_HX1), which we termed MeKL-chip. The kinase-modification in combination with the addition of PEG have increased the ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing. MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs. MBD-enrichment was performed on 3 x retina and 3 x brain DNA samples from adult C57BL/6J mice on varied DNA quantities (250, 50, 25 and 10 ng). Samples were amplified using a modified ligation-mediated PCR and hybridized to custom NimbleGen 2.1M mouse arrays (Feinberg_MM8_Me_HX1)
2013-06-10 | E-GEOD-46683 | biostudies-arrayexpress
Project description:Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using MBD-enrichment are restricted to large (≥1 µg) DNA samples, limiting the analysis of small tissue samples. Here we present a technique which enables characterization of genome wide tissue-specific methylation patterns from nanogram quantities of DNA. We have developed a methodology utilizing MBD2b/MBD3L1-enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized NimbleGen 2.1M mouse tiling arrays (Feinberg_MM8_Me_HX1), which we termed MeKL-chip. The kinase-modification in combination with the addition of PEG have increased the ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing. MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs.
2013-06-10 | GSE46683 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2015-11-19 | E-GEOD-69279 | biostudies-arrayexpress