Abstract
SummaryGenome-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.
Highlights
DNA methylation is an epigenetic mechanism that is indispensable for animal development (Reik, 2007) and broadly relevant for plant biology (Law and Jacobsen, 2010)
DNA is digested with the restriction enzymes MspI and/or TaqI, which are insensitive to DNA methylation at the central CpG, and short size-selected restriction fragments are subjected to bisulfite sequencing (Figure 2A)
We generated representation bisulfite sequencing (RRBS) libraries for nine species. These libraries showed characteristic fragment length distributions, which reflect the distribution of CpG-rich repetitive elements in these species and which provide a convenient metric for assessing the quality of RRBS libraries prior to sequencing (Figure 2B)
Summary
DNA methylation is an epigenetic mechanism that is indispensable for animal development (Reik, 2007) and broadly relevant for plant biology (Law and Jacobsen, 2010). Altered DNA methylation patterns are ubiquitous in cancer (Baylin and Jones, 2011; Feinberg and Tycko, 2004), and they have been observed in numerous other diseases (Portela and Esteller, 2010; Robertson, 2005). Epigenome-wide association studies (EWASs) have emerged as a widely used paradigm for linking DNA methylation to environmental exposures and to diseases (Michels et al, 2013; Rakyan et al, 2011). A widely discussed hypothesis posits that epigenetic mechanisms provide a mechanistic link between exposures and diseases, contributing to the developmental origins of health and disease in humans (Gillman, 2005; Waterland and Michels, 2007). DNA methylation can be transgenerationally inherited at certain genomic loci (Feil and Fraga, 2011) and may contribute to species evolution (Jablonka and Raz, 2009)
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