SGER: Gene Enrichment Sequencing of the Maize Genome with Hypomethylated Partial Restriction Libraries

这个探索性研究项目的长期目标是测试一种新的方法，以玉米作为模式物种，对高等植物基因组中富含基因的部分进行测序。像许多其他高等植物基因组一样，玉米的核DNA主要由重复序列组成。这些重复序列大多是各种类型的转座因子，超过60%的核DNA由反转录转座子组成。玉米中的许多反转录转座子体积很大，序列高度相似，这使得在试图组装鸟枪基因组序列时很难区分同一家族的单个元素。为了避免逆转录转座子和其他无法组装的重复序列测序的低效率，人们提出了各种基因富集技术来提高霰弹枪序列方法的效率。该项目将测试一项新技术，该技术依赖于大多数或所有组织中大多数玉米反转录转座子在5‘- cg -3’和5‘- CNG-3’序列的胞嘧啶上被修饰（甲基化）的事实。该技术将使用对5‘-CG-3’甲基化敏感的限制性内切酶进行部分消化，以创建低甲基化部分限制性内切酶（HMPR）文库，该文库将允许对玉米基因组以及任何其他植物，动物或微生物基因组进行高效的鸟枪测序，这些基因组具有大量重复DNA，在5‘-CG-3’序列上进行5-甲基化。每个文库将使用两种不同的限制性内切酶（HpaII和HpyCH4IV）以甲基化依赖的方式识别和切割特定位点。使用这些库中的克隆将生成总共3600个序列。序列将与完整的植物基因组数据库进行比较，以查看基因，转座元件和细胞器DNA的频率。这些比较将决定富集技术的成功程度。克隆和生成的信息将提供给所有感兴趣的各方，为未来高等真核生物基因组的研究提供宝贵的资源和工具。

The long-term goal of this Small Grant for Exploratory Research project is to test a new approach to sequencing the gene-rich portions of higher plant genomes, using maize as the model species. Like many other higher plant genomes, the nuclear DNA of maize is primarily composed of repetitive sequences. Most of these repeated sequences are transposable elements of various types, with over 60% of the nuclear DNA composed of retrotransposons. Many of the retrotransposons in maize are large in size and highly similar in sequence, making it very difficult to distinguish between individual elements of the same family when trying to assemble a shotgun genome sequence. In order to avoid the inefficiency of sequencing retrotransposons and other repeats that cannot be assembled, various gene-enrichment technologies have been proposed to improve the efficacy of a shotgun sequence approach. This project will test a new technology that relies on the fact that most maize retrotransposons are modified (methylated) at cytosines in the sequences 5'-CG-3' and 5'- CNG-3' in most or all tissues. This technique will use partial digestion with restriction enzymes that are sensitive to 5'-CG-3' methylation to create hypomethylated partial restriction (HMPR) libraries that will permit high efficiency shotgun sequencing of the maize genome, and of any other plant, animal or microbial genome that has a large component of repetitive DNA that is 5-methylated at 5'-CG-3' sequences.Six libraries each will be produced using two different restriction enzymes (HpaII and HpyCH4IV) that recognize and cut specific sites in a methylation-dependent manner. A total of 3600 sequences will be generated using clones from these libraries. Sequences will be compared against the full plant genome databases to see the frequency of genes, transposable elements, and organellar DNA. These comparisons will determine the degree to which the enrichment technology has been successful. The clones and information generated will be available to all interested parties, providing a valuable resource and tool for the future study of higher eukaryotic genomes.