Techniques for Efficient Finishing and Physical Linkage of Gene-Enriched Shotgun Sequences

该概念验证项目的总体目标是开发玉米基因组资源，将其他正在进行的项目（DBI-0221536 [PI：Schubert]和DBI-0211851 [PI：Messing]）的序列连接在一起。 玉米基因组的长度约为34亿个碱基对（MB），但基因仅覆盖约150-450 MB或基因组的20%。初步结果表明，剩余的80%的基因组包含重复的DNA，其中大部分是高度甲基化的。这些基因被认为是分布在整个重复DNA海洋中的小岛。 目前正在评估分离和测序基因的方法，因为目前处理整个基因组是不可行的或不具成本效益的。 该项目的第一部分将使用Hypomethylated Partial Restriction（HMPR）文库来桥接基因内的小甲基化区域，这些区域在基因富集方法中丢失，这些方法基于甲基化过滤掉重复DNA。 HMPR序列可以允许通过桥接这些缺口来组装连续的基因序列。 该项目的第二部分是生成和测序甲基化跨越连接库（MSLL），这将提供一个从一个基因岛到下一个基因岛的甲基化重复DNA海洋的桥梁。将从两端对HMPR和MSLL文库克隆进行测序，并将序列与可用的玉米基因组序列组装。 最终的结果将有助于建立一个玉米基因组图谱中基因丰富的序列框架，所有的序列信息将立即发布到GenBank。 将在http://www.genome.arizona.edu上创建一个基于网络的显示，以提供对项目结果的访问。

The overall goal of this proof-of-concept project is to develop genomic resources for maize that will link together sequences available from other ongoing projects (DBI-0221536 [PI: Schubert] and DBI-0211851 [PI: Messing]). The maize genome is approximately 3,400 million base pairs (MB) in length, yet the genes only cover about 150-450 MB or 20% of the genome. Preliminary results indicate the remaining 80% of the genome comprises repetitive DNAs, much of which is highly methylated. The genes are thought to be distributed as small islands throughout the sea of repetitive DNA. Methods to isolate and sequence the genes are currently being evaluated since it is not feasible or cost-effective to tackle the whole genome at this time. The first part of this project will use HypoMethylated Partial Restriction (HMPR) libraries to bridge small methylated regions within genes that are lost in gene enrichment methods that filter out repetitive DNA based on methylation. The HMPR sequences could allow contiguous gene sequences to be assembled by bridging these gaps. The second part of this project is to generate and sequence Methylation Spanning Linker Libraries (MSLLs) that will provide a bridge over the sea of methylated repetitive DNA from one gene island to the next. The HMPR and MSLL library clones will be sequenced from both ends and the sequences assembled with the available maize genomic sequence. The end results should contribute to the development of a scaffold of gene-rich sequences positioned across the maize genome map.All sequence information will be released immediately to GenBank. A web-based display will be created at http://www.genome.arizona.edu to provide access to the project results.