A new Agilent 15K cross strain P. falciparum microarray optimized for the transcriptome analysis of Indian P. falciparum isolates. Plasmodium falciparum

Genomic variation is an inherent phenomena observed among members of same species belonging to different geographical locations. In case of P. falciparum, an apicomplexan protozoan parasite, its 22.8 MB nuclear genome is known to display vast genetic diversity in the subtelomeric compartments having but not exclusively variant gene families like var, rifins and stevors and examples in other elements of the genome have recently been documented. Microarrays, relies solely on the genomic sequence information to capture the relevant transcript abundance and needs to consider these variations into account for revealing true transcriptional variation.Here, we describe the designing strategy of a custom P. falciparum 15K array using Agilent platform to study the transcriptome of Indian field isolates for which genome sequence information is limited. Array contains probes representing genome sequence of two distinct geographical isolates (i.e 3D7 and HB3) and subtelomeric var gene sequence of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts by performing a 244K array experiment representing multiple probes per gene/transcript. Array performance was evaluated and validated using RNA materials from P. falciparum clinical isolates obtained directly from patients with differing clinical conditions due to malaria infection.Due to pre probe screening large percentage (91 %) of the represented transcripts could be detected from Indian P. falciparum isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. Overall design: Plasmodium falciparum isolates were collected from patients (n=13) with differing clinical conditions. The patients exhibited symptoms categorized as uncomplicated (n=6) or complicated malaria (n=7). Criteria for determination of complicated disease were based on World Health Organization year 2000 guidelines. Microarray array based transcriptional profiling was carried out to evaluate the performance of the array.

基因组变异是分布于不同地理区域的同一物种成员间普遍存在的固有现象。就恶性疟原虫（Plasmodium falciparum）——一种顶复门原生动物寄生虫——而言，其22.8 Mb的核基因组已被证实于亚端粒区展现出极高的遗传多样性，该区域包含但不限于var、rifin及stevor等变异基因家族，且近期已有研究在基因组其他元件中报道了类似的多样性案例。微阵列（Microarrays）仅依赖基因组序列信息来捕获相关转录本的丰度，因此在揭示真实的转录变异时，必须将这些基因组变异纳入考量范畴。本研究详细阐述了基于安捷伦（Agilent）平台定制的恶性疟原虫15K微阵列的设计策略，该阵列用于研究基因组序列信息有限的印度野外分离株的转录组。该阵列的探针涵盖两类不同地理分离株（即3D7与HB3）的基因组序列，以及第三株在培养条件下可黏附的分离株IT4的亚端粒var基因序列。阵列探针的筛选基于其检测转录本的效能，通过开展244K阵列实验完成，每个基因/转录本对应多个探针。研究使用直接从疟疾感染且临床症状各异的患者体内获取的恶性疟原虫临床分离株的RNA样本，对该阵列的性能进行了评估与验证。由于预先开展了探针筛选，本阵列可从印度恶性疟原虫分离株中检测到占比高达91%的目标转录本。重复探针以及代表同一基因的多个探针之间呈现出完美的相关性，表明探针性能优异。由于纳入了代表HB3株转录本的独特探针，本阵列还可检测到额外的转录本。通过针对三种地理分布各异菌株的变异表面抗原（Variant Surface Antigen, VSA）基因优化设计的探针，成功检测到了VSA转录本。整体实验设计：从13名临床症状各异的疟疾患者体内分离得到恶性疟原虫菌株，其中6名患者表现为单纯性疟疾，7名为重症疟疾。重症疾病的判定标准参照世界卫生组织2000年版指南。本研究通过基于微阵列的转录谱分析，对该阵列的性能进行了评估。