Subudhi Amit Kumar, Boopathi P A, Middha Sheetal, Acharya Jyoti, Rao Sudha Narayana, Mugasimangalam Raja C, Sirohi Paramendra, Kochar Sanjay K, Kochar Dhanpat K, Das Ashis
Department of Biological Sciences, Birla Institute of Technology and Science (BITS), Pilani, Rajasthan, India.
Department of Medicine, S.P. Medical College, Bikaner, Rajasthan, India.
Genom Data. 2016 Jul 20;9:118-25. doi: 10.1016/j.gdata.2016.07.006. eCollection 2016 Sep.
Malarial parasite P. falciparum, an apicomplexan protozoan has a 23.3 MB nuclear genome and encodes ~ 5600 transcripts. The genetic diversity of the parasite within and across geographical zones is a challenge to gene expression studies which are essential for understanding of disease process, outcome and developing markers for diagnostics and prognostics. Here, we describe the strategy involved in designing a custom P. falciparum 15K array using the Agilent platform and Genotypic's Right Design methodology to study the transcriptome of Indian field isolates for which genome sequence information is limited. The array contains probes representing genome sequences of two distinct geographical isolates (i.e. 3D7 and HB3) and sub-telomeric var gene sequences 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 through a 244K array experimentation. Array performance for the 15K array, was evaluated and validated using RNA materials from P. falciparum clinical isolates. A large percentage (91%) of the represented transcripts was detected from Indian P. falciparum patient 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. The 15K cross strain P. falciparum array has shown good efficiency in detecting transcripts from P. falciparum parasite samples isolated from patients. The low parasite loads and presence of host RNA makes arrays a preferred platform for gene expression studies over RNA-Seq.
恶性疟原虫是一种顶复门原生动物，其核基因组大小为23.3兆碱基，编码约5600个转录本。该寄生虫在地理区域内和区域间的遗传多样性对基因表达研究构成了挑战，而基因表达研究对于理解疾病过程、结果以及开发诊断和预后标志物至关重要。在此，我们描述了使用安捷伦平台和Genotypic公司的Right Design方法设计定制的恶性疟原虫15K芯片的策略，以研究基因组序列信息有限的印度野外分离株的转录组。该芯片包含代表两种不同地理分离株（即3D7和HB3）基因组序列以及已知在培养条件下具有黏附性的第三种分离株（IT4）的亚端粒var基因序列的探针。芯片中的探针是基于它们通过244K芯片实验检测转录本的效率而选择的。使用来自恶性疟原虫临床分离株的RNA材料对15K芯片的性能进行了评估和验证。从印度恶性疟原虫患者分离株中检测到了很大比例（91%）的代表性转录本。代表同一基因的重复探针和多个探针之间显示出完美的相关性，表明探针性能良好。由于包含了代表HB3菌株转录本的独特探针，还检测到了额外的转录本。通过代表三种地理上不同菌株VSA基因的优化探针检测到了变异表面抗原（VSA）转录本。15K跨菌株恶性疟原虫芯片在检测从患者分离的恶性疟原虫寄生虫样本的转录本方面显示出良好的效率。低寄生虫载量和宿主RNA的存在使得芯片成为比RNA测序更适合基因表达研究的平台。
