References
Extensive promoter DNA hypermethylation and hypomethylation is associated with aberrant microRNA expression
in chronic lymphocytic leukemia
Quality control and single nucleotide resolution analysis of methylated DNA
immunoprecipitation products
Auto MeDIP-seq: A high-throughput, whole genome, DNA methylation assay
PML-RARa/RXR Alters the Epigenetic Landscape in Acute Promyelocytic Leukemia
Whole-genome DNA methylation profiling using MethylCap-seq
Quantitative comparison of genome-wide DNAmethylation mapping technologies
1. Automated immunoprecipitation of methylated DNA (MeDIP)
Automated immunoprecipitation of methylated DNA (MeDIP) using the Diagenode IP star is currently applied to several large scale cancer methylome projects some of them under the coordination of the International Cancer Genome Consortium at the Centre National de Génotypage (Evry, France). Automated MeDIP has successfully been coupled to promoter and CpG island tiling arrays as well as second generation sequencing. The automation of the immunoprecipitation has been shown to significantly increase the signal to noise ratio permitting a more rapid and reliable identification of differentially methylated candidate genes as assessed by high resolution quantitative pyrosequencing.
Dr Jörg Tost Centre National de Génotypage, Evry, France
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2. Automating whole-genome DNA methylation analyses
To automate the hands-on aspect of Methylated DNA Immunoprecipitation (MeDIP) - "Auto MeDIP" & "Auto ChIP" - Diagenode recently introduced a robotic liquid handler. We are currently using this machine to perform a series of custom assays with qPCR, microarrays and, ultimately, Illumina Solexa 2nd-generation sequencing. The Auto MeDIP workflow qualitatively distinguishes between methylated and unmethylated fragments. Future work: we aim test the Auto MeDIP workflow with Illumina sequencing using both control DNA and, as a proof-of-principle, experimental samples. These experiments will help fine-tune the methylation-scoring algorithms for use in future studies.
Dr Stephan Beck University College London London, WC1E 6BT, United Kingdom
Contact Person: Lee Butcher
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3. MethylCap-sequencing (Methyl binding domain - sequencing)
DNA methylation is the most stable epigenetic mark that is strongly associated with cancer. Genome-wide mapping of DNA methylation associated with human cancers provides a readout of their cancer epigenomes. The general expection is that catalogues of the differentially methylated regions will be of high diagnostic and/or prognostic value. Most studies performed thus far have been limited to proximal promoters and CpG islands. It is possible that genomic regions outside of promoters and CpG islands are also targets for differential methylation in a "cancer-type" specific manner. A bottleneck in the genome-wide analysis of DNA methylation has been the lack of methods amenable for genome-scale analysis. Here we present a strategy based on capturing of methylated DNA fragments using an MBD protein (MethylCap), followed by deep sequencing that allows to interrogate the entire genome. The use of automated MethylCap guarentees high reproducibility and high-throughput in generating whole-genome DNA methylation profiles of human (cancer) genomes.
Project start: April 2009 (still in progress)
Dr Henk Stunnenberg Nijmegen Center for Molecular Life Sciences Nijmegen, The Netherlands
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