title: Differential DNA Methylation Analysis without a Reference Genome.
abstract: 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 DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of 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, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified 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.
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.
authors.forename: Johanna
authors.initials: J
authors.lastname: Klughammer
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Datlinger
authors.affiliation: Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, 1090 Vienna, Austria.
authors.forename: Dieter
authors.initials: D
authors.lastname: Printz
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Sheffield
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.
authors.forename: Matthias
authors.initials: M
authors.lastname: Farlik
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.
authors.forename: Johanna
authors.initials: J
authors.lastname: Hadler
authors.affiliation: Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, 1090 Vienna, Austria.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Fritsch
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria; Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria; Max Planck Institute for Informatics, 66123 Saarbrücken, Germany. Electronic address: cbock@cemm.oeaw.ac.at.
authors.forename: Christoph
authors.initials: C
authors.lastname: Bock
mesh_terms: D000818:Animals; D001773:Blood Cells / Q000378:metabolism; D002347:Carps; D002417:Cattle; D002874:Chromosome Mapping; D018899:CpG Islands; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D016678:Genome; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012984:Software
chemical_list: D004247:DNA
keywords: DNA methylation; RRBS; bisulfite sequencing; comparative genomics; computational epigenetics; cross-species comparison; differential methylation analysis; non-model organisms; reference genome independent analysis; vertebrate genomes