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来测序高等植物基因组的基因丰富的部分，使用玉米作为模式物种。与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。这些重复序列中的大多数是各种类型的转座因子，其中超过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尺寸很大，序列高度相似，使得在试图组装鸟枪基因组序列时很难区分同一家族的各个元件。为了避免测序反转录转座子和其他不能组装的重复序列的效率低下，已经提出了各种基因富集技术来提高鸟枪测序方法的效率。该项目将测试一项新技术，该技术依赖于大多数玉米逆转录转座子在大多数或所有组织中的5 '-CG-3'和5 '-CNG-3'序列中的胞嘧啶处被修饰（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酶的部分消化来产生低甲基化部分限制性（HMPR）文库，其将允许玉米基因组和任何其它植物的高效鸟枪测序，动物或微生物基因组，其具有在5 '-CG-1处5-甲基化的大部分重复DNA。使用两种不同的限制性酶（HpaII和HpyCH 4 IV）产生六个文库，所述限制性酶以甲基化依赖性方式识别和切割特定位点。使用来自这些文库的克隆将产生总共3600个序列。将序列与完整的植物基因组数据库进行比较，以查看基因，转座因子和细胞器DNA的频率。这些比较将确定浓缩技术的成功程度。克隆和产生的信息将提供给所有感兴趣的各方，为高等真核生物基因组的未来研究提供了宝贵的资源和工具。

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.