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      Variations in 5-methylcytosine and 5-hydroxymethylcytosine among human brain, blood, and saliva using oxBS and the Infinium MethylationEPIC array

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          Abstract

          Investigating 5-methylcytosine (5mC) has led to many hypotheses regarding molecular mechanism underlying human diseases and disorders. Many of these studies, however, utilize bisulfite conversion alone, which cannot distinguish 5mC from its recently discovered oxidative product, 5-hydroxymethylcytosine (5hmC). Furthermore, previous array-based technologies do not have the necessary probes to adequately investigate both modifications simultaneously. In this manuscript, we used technical replicates of DNA from human brain, human blood, and human saliva, in combination with oxidative bisulfite conversion and Illumina’s Infinium MethylationEPIC array, to analyze 5mC and 5hmC at more than 650 000 and 450 000 relevant loci, respectively, in the human genome. We show the presence of loci with detectable 5mC and 5hmC to be equally distributed across chromosomes and genomic features, while also being present in genomic regions with transcriptional regulatory properties. We also describe 2528 5hmC sites common across tissue types that show a strong association with immune-related functions. Lastly, in human brain, we show that 5hmC accounts for one-third of the total signal from bisulfite-converted data. As such, not only do our results confirm the efficacy and sensitivity of pairing oxidative bisulfite conversion and the EPIC array to detect 5mC and 5hmC in all three tissue types, but they also highlight the importance of dissociating 5hmC from 5mC in future studies related to cytosine modifications.

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          Most cited references 34

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          Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.

          DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference-mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.
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            Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays.

            The recently released Infinium HumanMethylation450 array (the '450k' array) provides a high-throughput assay to quantify DNA methylation (DNAm) at ∼450 000 loci across a range of genomic features. Although less comprehensive than high-throughput sequencing-based techniques, this product is more cost-effective and promises to be the most widely used DNAm high-throughput measurement technology over the next several years. Here we describe a suite of computational tools that incorporate state-of-the-art statistical techniques for the analysis of DNAm data. The software is structured to easily adapt to future versions of the technology. We include methods for preprocessing, quality assessment and detection of differentially methylated regions from the kilobase to the megabase scale. We show how our software provides a powerful and flexible development platform for future methods. We also illustrate how our methods empower the technology to make discoveries previously thought to be possible only with sequencing-based methods. http://bioconductor.org/packages/release/bioc/html/minfi.html. khansen@jhsph.edu; rafa@jimmy.harvard.edu Supplementary data are available at Bioinformatics online.
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              Orchestrating high-throughput genomic analysis with Bioconductor.

              Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors.
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                Author and article information

                Journal
                Biol Methods Protoc
                Biol Methods Protoc
                biomap
                Biology Methods & Protocols
                Oxford University Press
                2396-8923
                March 2016
                27 December 2016
                27 December 2016
                : 1
                : 1
                : 1-8
                Affiliations
                [1 ]McGill Group for Suicide Studies, Douglas Mental Health University Institute, Department of Psychiatry, McGill University, Montreal, Quebec, Canada;
                [2 ]Canadian Centre for Computational Genomics, McGill University, Montreal, Quebec, Canada;
                [3 ]McGill University and Genome Quebec Innovation Centre, McGill University, Montreal, Quebec, Canada,
                [4 ]Department of Human Genetics, McGill University, Montreal, Quebec, Canada
                Author notes
                [* ]Correspondence address. McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, 6875 boul. LaSalle, Verdun, Quebec H4H 1R3, Canada. Tel: +1-514-761-6131 ext. 2369; Fax: +1-514-762-3023; E-mail: gustavo.turecki@ 123456mcgill.ca
                Article
                bpw002
                10.1093/biomethods/bpw002
                7164292
                © The Author 2016. Published by Oxford University Press. All rights reserved.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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                Pages: 8
                Product
                Funding
                Funded by: Canadian Institute of Health Research
                Award ID: MOP93775, MOP11260, MOP119429
                Award ID: MOP119430
                Funded by: National Institutes of Health
                Award ID: 1R01DA033684
                Funded by: Pfizer Canada
                Funded by: Fonds de recherche du Québec - Santé
                Funded by: Genome Canada
                Funded by: CIHR Frederick Banting and Charles Best Doctoral fellowship
                Funded by: Fondation Fyssen
                Funded by: Canadian Institutes of Health Research
                Funded by: American Foundation for Suicide Prevention
                Categories
                Methods Manuscript

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