Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Methylation profiling of human samples using MeDIP-seq method

ABSTRACT: Aim: We aim to compare current (MeDIP-seq), new (Illumina Infinium 450K BeadChip) and future (PacBio) methods for whole genome DNA methylation analysis. As the interest in determination of disease methylation profiles increases, the scope, advantages and limitations of these methods requires assessment. There are key questions to answer and specific challenges to overcome. For example, how much detail/resolution is sufficient to identify regions of differential methylation and regions of biological/medical significance within a sample? How much coverage of the genome is required for accurate methylation analysis? Is it important to confirm which regions of the genome are unmethylated in addition to focusing on those that are methylated? Loss of methylation may be of equal importance within the cell since this may also contribute to disease pathogenesis. A multi-method (affinity enrichment/bisulphite-conversion based/direct sequencing of methyl-cytosine) and technology platform (Illumina HiSeq/PacBio/Illumina Infinium BeadChip) comparison will enable us to determine the strengths and weakness of each method. We propose to compare four methods using two DNA samples from the Coriell Institute for Cell Repository to assess both current and future capabilities for whole genome methylation analysis in parallel: A) MeDIP-seq using Illumina HiSeq B) Illumina Infinium HumanMethylation 450K BeadChip and C) whole genome methylation sequencing using PacBio. Existing single molecule deep bisulphite sequencing data generated previously from these same samples at the WTSI for targeted regions (30-40 genes) on the human X chromosome will be used to assess performance of each method. The methods selected for this study will generate data covering a range of resolutions from a whole genome scan to array (target defined) resolution and up to single base pair, single molecule resolution; the highest level of detail possible with methods currently available.Samples: DNA from sibling pair GM01240 (female) and GM01240 (male).Requirements: Both samples will be analysed using;A.MeDIP-seq using Illumina HiSeq (one HiSeq lane, 75bp paired end, per sample) B.Illumina Infinium HumanMethylation 450K BeadChipWe are expecting a potentially unnecessary high coverage using one HiSeq lane per sample. However, for the MeDIP procedure we do not have a multiplexing procedure in place. Our requirements for PacBio sequencing have been discussed with and will be supported by the Sequencing Technology Development group.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

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PROVIDER: E-ERAD-139 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aim: We aim to compare current (MeDIP-seq), new (Illumina Infinium 450K BeadChip) and future (PacBio) methods for whole genome DNA methylation analysis. As the interest in determination of disease methylation profiles increases, the scope, advantages and limitations of these methods requires assessment. There are key questions to answer and specific challenges to overcome. For example, how much detail/resolution is sufficient to identify regions of differential methylation and regions of biological/medical significance within a sample? How much coverage of the genome is required for accurate methylation analysis? Is it important to confirm which regions of the genome are unmethylated in addition to focusing on those that are methylated? Loss of methylation may be of equal importance within the cell since this may also contribute to disease pathogenesis. A multi-method (affinity enrichment/bisulphite-conversion based/direct sequencing of methyl-cytosine) and technology platform (Illumina HiSeq/PacBio/Illumina Infinium BeadChip) comparison will enable us to determine the strengths and weakness of each method. We propose to compare four methods using two DNA samples from the Coriell Institute for Cell Repository to assess both current and future capabilities for whole genome methylation analysis in parallel: A) MeDIP-seq using Illumina HiSeq B) Illumina Infinium HumanMethylation 450K BeadChip and C) whole genome methylation sequencing using PacBio. Existing single molecule deep bisulphite sequencing data generated previously from these same samples at the WTSI for targeted regions (30-40 genes) on the human X chromosome will be used to assess performance of each method. The methods selected for this study will generate data covering a range of resolutions from a whole genome scan to array (target defined) resolution and up to single base pair, single molecule resolution; the highest level of detail possible with methods currently available. Samples: DNA from sibling pair GM01240 (female) and GM01240 (male). Requirements: Both samples will be analysed using; A. MeDIP-seq using Illumina HiSeq (one HiSeq lane, 75bp paired end, per sample) B. Illumina Infinium HumanMethylation 450K BeadChip We are expecting a potentially unnecessary high coverage using one HiSeq lane per sample. However, for the MeDIP procedure we do not have a multiplexing procedure in place. Our requirements for PacBio sequencing have been discussed with and will be supported by the Sequencing Technology Development group.

Project description:This study focuses on epigenetic reprogramming in the mouse germ line: DNA methylation marks, including those of imprinted genes, are thought to be erased between E11.5 and E13.5 in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. DNA methylation patterns are then re-established during the de novo methylation phase in the male germ line several days later (around E15.5) and in the female germ line after birth during adult life. Epigenetic reprogramming in PGCs is poorly understood mainly because of the technical challenges that arise from very low cell numbers in the embryo. The aim of this study is to create genome-wide maps of DNA methylation patterns of male and female PGCs at crucial time points during epigenetic reprogramming and to investigate the changes in those profiles on a single-gene level. We have already successfully prepared and sequenced the first set of BS-Seq libraries of PGCs with Illumina's GAIIx platform. From this, we gained significant insight into the global DNA methylation levels and methylation patterns over multy-copy-loci such as repeat elements. In order further enhance our analysis and finish this project for publication, it is crucial to increase the genomic coverage of our datasets. This will also allow us to link the BS-Seq data with other MeDIP-Seq and RNA-Seq datasets that have already been created in this study.Protocol: Input DNA was sonicated using a Bioruptor UCD-200 (Diagenode) to a final size distribution of 300bp 1000bp. End-repair and A-tailing were performed with the NEBNext DNA Sample Prep Master Mix Set 1. Illuminas Early Access Methylation Adaptor Oligo Kit was used for the adapter ligation. The adapter-ligated DNA was treated with sodium-bisulfite using the Imprint DNA Modification Kit from Sigma-Aldrich according to the manufacturers instructions for the two-step protocol. After the clean up, the bisulfite-treated DNA was amplified using PfuTurbo Cx Hotstart DNA Polymerase from Agilent with 18 cycles of amplification. The entire PCR reaction was run on a 1.5% agarose TBE gel and size selection was performed for DNA fragments between 200bp 250bp. DNA from the excised gel piece was purified with the Qiagen Gel Extraction Kit.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

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Methylation profiling of human samples using MeDIP-seq method

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