Wischnitzki Elisabeth, Sehr Eva Maria, Hansel-Hohl Karin, Berenyi Maria, Burg Kornel, Fluch Silvia
AIT Austrian Institute of Technology GmbH, Konrad-Lorenzstreet 24, 3430 Tulln, Austria.
Biomed Res Int. 2015;2015:570568. doi: 10.1155/2015/570568. Epub 2015 Sep 3.
Genome assembly remains a challenge for large and/or complex plant genomes due to their abundant repetitive regions resulting in studies focusing on gene space instead of the whole genome. Thus, DNA enrichment strategies facilitate the assembly by increasing the coverage and simultaneously reducing the complexity of the whole genome. In this paper we provide an easy, fast, and cost-effective variant of MRE-seq to obtain a plant's hypomethylome by an optimized methyl filtration protocol followed by next generation sequencing. The method is demonstrated on three plant species with knowingly large and/or complex (polyploid) genomes: Oryza sativa, Picea abies, and Crocus sativus. The identified hypomethylomes show clear enrichment for genes and their flanking regions and clear reduction of transposable elements. Additionally, genomic sequences around genes are captured including regulatory elements in introns and up- and downstream flanks. High similarity of the results obtained by a de novo assembly approach with a reference based mapping in rice supports the applicability for studying and understanding the genomes of nonmodel organisms. Hence we show the high potential of MRE-seq in a wide range of scenarios for the direct analysis of methylation differences, for example, between ecotypes, individuals, within or across species harbouring large, and complex genomes.
由于大型和/或复杂植物基因组存在大量重复区域,基因组组装仍然是一项挑战,这导致研究主要集中在基因空间而非整个基因组。因此,DNA富集策略通过增加覆盖度并同时降低整个基因组的复杂度来促进组装。在本文中,我们提供了一种简单、快速且经济高效的MRE-seq变体,通过优化的甲基过滤方案结合下一代测序来获得植物的低甲基化组。该方法在三种已知具有大型和/或复杂(多倍体)基因组的植物物种上得到了验证:水稻、欧洲云杉和藏红花。所鉴定的低甲基化组显示出基因及其侧翼区域的明显富集以及转座元件的明显减少。此外,还捕获了基因周围的基因组序列,包括内含子以及上下游侧翼中的调控元件。在水稻中,通过从头组装方法获得的结果与基于参考图谱的映射结果高度相似,这支持了该方法在研究和理解非模式生物基因组方面的适用性。因此,我们展示了MRE-seq在广泛场景下直接分析甲基化差异的巨大潜力,例如在生态型、个体之间,以及在拥有大型和复杂基因组的物种内部或物种之间。