High-resolution hybridization-based genotyping in Plasmodium falciparum

描述（由申请人提供）：最近对几种致命的疟疾寄生虫恶性疟原虫进行了测序，为该物种的DNA多样性提供了丰富的比较数据库。这些庞大的信息可能会激发令人兴奋的新研究；然而，无法在自然种群的全基因组规模上轻松测定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，如拷贝数多态性，索引，甚至SNPs。顶复合体恶性疟原虫基因组相对较小（23mb），在人红细胞中是单倍体，使其成为CGH基因分型的极好候选。缺点是该物种的A - T丰富度（80%），这是一个挑战，可以通过使用较长的寡核苷酸探针来弥补相对较低的熔化温度，同时也减少了基因组中其他位点交叉杂交的可能性。使用无掩膜光刻技术的CGH平台对于基于杂交的基因分型和重测序具有很大的前景，因为它使用高密度格式的更长和可变长度的探针，并且允许经济有效的，按需的单芯片设计；因此，不同的芯片配置可以评估其检测snp的能力。我们使用更长的探针对恶性疟原虫进行了CGH，并成功地鉴定了多态性谱，包括单碱基变化。此外，我们已经证明探针参数的变化会显著影响这种能力。本研究的目的是对恶性疟原虫基因分型芯片进行系统优化。我们将使用Broad/Harvard/MIT测序联盟编译的SNP数据库来直接优化探针配置。具体目标1：利用HB3和3D7菌株dna与一系列由跨越已知SNP的单碱基平铺探针组成的芯片共杂交来优化SNP检测探针。具体目标2。利用优化后的探针选择全基因组单特征多态性（snp和indel），包括序列重复性增加的区域，并直接对twp 48kb区域进行重测序。特定目标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后提供，以促进社区快速使用这项技术。疟疾每年导致非洲200多万儿童死亡，全球感染人数超过5亿。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为该物种的遗传多样性提供了一个广阔的视角；然而，研究人员没有办法在大量人群中分析这种差异。我们的建议旨在开发一个基因分型微阵列平台，促进关联研究和单倍型定位，以发现疟疾寄生虫耐药性和毒力的遗传决定因素。这一知识将导致新的途径来攻击这种疾病。

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.