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A custom genome-wide Plasmodium vivax 8x15K microarray for expression profiling of Indian clinical isolates showing complicated malaria

ABSTRACT: High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n=14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n=85) present in the arrays showed perfect correlation (r2=0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r≥0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates.

ORGANISM(S): Plasmodium vivax

PROVIDER: GSE55644 | GEO | 2016/10/18

SECONDARY ACCESSION(S): PRJNA240292

REPOSITORIES: GEO

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Project description: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. 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.

Project description:Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is very little understood. Emerging evidences of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating this parasite directly from the infected patients is the most feasible way to study its biology and any pathogenic mechanisms which may exist. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. However, the mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of natural antisense transcripts (NATs) in P. falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed strand specific microarray. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense transcript. Our data also shows condition specific expression patterns of varying S and AS transcript levels. Genes with AS transcripts enrich to various biological processes. This is the first report detailing the presence of NATs from clinical isolates of P. vivax. The data suggests differential regulation of gene expression in diverse clinical conditions and would lead to future detailed investigations of genome regulation. Plasmodium vivax isolates were collected from patients (n = 8) with differing clinical conditions.The patients exhibited symptoms categorized as un-complicated (n =1) or complicated malaria (n = 7). Criteria for determination of complicated disease were based on World Health Organization year 2010 guidelines. Microarray array based transcriptional profiling was carried out to detect prevalence of natural antisense transcripts.

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A custom genome-wide Plasmodium vivax 8x15K microarray for expression profiling of Indian clinical isolates showing complicated malaria

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