High-resolution hybridization-based genotyping in Plasmodium falciparum

描述（由申请人提供）：几种致命的疟疾寄生虫，恶性疟原虫，最近已经测序，提供了丰富的比较数据库的DNA多样性，为这个物种。这一巨大的信息可能会刺激令人兴奋的新研究;然而，无法在自然种群中的全基因组规模上轻松分析这种变异是一个重大障碍。高密度比较基因组杂交（CGH）可用于扫描基因组的变异，例如拷贝数多态性、插入/缺失甚至SNP。顶复门恶性疟原虫基因组相对较小（23 Mb），在人类红细胞中为单倍体，使其成为CGH基因分型的绝佳候选者。一个缺点是该物种的A-T丰富度（80%），这是一个可以通过使用较长的寡核苷酸探针来克服的挑战，以补偿相对较低的解链温度，同时还降低来自基因组中其他基因座的交叉杂交潜力的可能性。使用无掩模光刻技术的CGH平台对于基于杂交的基因分型和重新测序具有很大的希望，因为它以高密度格式使用更长和可变长度的探针，并且允许具有成本效益的、按需的单芯片设计;因此，可以评估不同的芯片配置检测SNP的能力。我们已经使用较长的探针对恶性疟原虫进行了CGH，并成功地鉴定了一系列多态性，包括单碱基变化。此外，我们已经证明，探针参数的变化显着影响这种能力。本研究的目的是系统地优化恶性疟原虫基因分型芯片。我们将通过使用Broad/哈佛/麻省理工学院测序联盟编译的SNP数据库来直接优化我们的探针配置。具体目标1：利用HB 3和3D 7株DNA与一系列由跨越已知SNPs的单碱基平铺探针组成的芯片共杂交，优化用于SNP检测的探针。具体目标2。将优化的探针应用于选择全基因组的单特征多态性（SNP和indel），包括序列重复性增加的区域，并直接重测序两个48 kb区域。具体目标3：将这些设计参数扩展到所有已知的多态性，并开发定制的恶性疟原虫基因分型芯片。我们的原始数据，分析和芯片设计将在生成时提供，以促进社区快速使用这项技术。疟疾每年在非洲造成200多万儿童死亡，在全世界感染5亿多人。布罗德研究所最近完成的恶性疟原虫比较基因组测序项目提供了该物种遗传多样性的广阔前景;然而，研究人员没有办法在大种群中分析这种变异。我们的建议旨在开发一个基因分型微阵列平台，这将有助于关联研究和单倍型定位，以找到疟原虫耐药性和毒力的遗传决定因素。这一知识将导致对这种疾病的攻击的新途径。

DESCRIPTION (provided by applicant): Several strains of the lethal malaria parasite, Plasmodium falciparum, have recently been sequenced, providing a rich comparative database of DNA diversity for this species. This vast information could spur exciting new research; however, the inability to easily assay this variation on a whole-genome scale in natural populations is a significant hindrance. High-density comparative genome hybridization (CGH) can be used to scan genomes for variation such as copy number polymorphisms, indels, and even SNPs. The apicomplexan P. falciparum genome is relatively small (23 Mb) and haploid in human red blood cells, making it a superb candidate for CGH genotyping. A drawback is the A - T richness of this species (80%), a challenge that can be overcome by using longer oligonucleotide probes to compensate for the relatively low melting temperature while also reducing the likelihood of cross-hybridization potential from other loci in the genome. A CGH platform that uses a maskless photolithography technology holds great promise for hybridization-based genotyping and re-sequencing because it uses longer and variable length probes in a high density format, and allows for cost-effective, as-needed, single chip designs; consequently, different chip configurations can be evaluated for their ability to detect SNPs. We have performed CGH for P. falciparum using longer probes and have had success identifying a spectrum of polymorphisms, including single base changes. Moreover, we have demonstrated that variations in probe parameters significantly influence this capacity. The goal of this proposal is to systematically optimize a P. falciparum genotyping chip. We will do this by using the SNP database compiled by the Broad/Harvard/MIT sequencing consortium to directly optimize our probe configurations. Specific Aim 1: To optimize probes for SNP detection using co- hybridization of the HB3 and 3D7 strain DNAs to a series of chips consisting of single- base tiled probes spanning known SNPs. Specific Aim 2. To apply the optimized probes to select single feature polymorphisms (SNPs and indels) genome-wide, including regions of increasing sequence repetitiveness, and to directly re-sequence twp 48kb regions. Specific Aim 3: To extend these design parameters to all known polymorphisms and develop a custom P. falciparum genotyping chip. Our raw data, analysis, and chip designs will be made available as generated to facilitate rapid community use of this technology. Malaria kills more than 2 million kids in Africa and infects more than 500 million people worldwide each year. The recently completed Plasmodium falciparum comparative genome sequencing project at the Broad Institute provides a vast view of genetic diversity of this species; however, there is no way for researchers to assay this variation in large populations. Our proposal aims to develop a genotyping microarray platform that will facilitate association studies and haplotype mapping to find the genetic determinants of malaria parasite drug resistance and virulence. This knowledge will lead to new avenues of attack against this disease.