Simultaneous genome-wide gene expression and transcript isoform profiling in the human malaria parasite

<div><p>Gene expression DNA microarrays have been vital for characterizing whole-genome transcriptional profiles. Nevertheless, their effectiveness relies heavily on the accuracy of genome sequences, the annotation of gene structures, and the sequence-dependent performance of individual probes. Currently available gene expression arrays for the malaria parasite <i>Plasmodium falciparum</i> rely on an average of 2 probes per gene, usually positioned near the 3′ end of genes; consequently, existing designs are prone to measurement bias and cannot capture complexities such as the occurrence of transcript isoforms arising from alternative splicing or alternative start/ stop sites. Here, we describe two novel gene expression arrays with exon-focused probes designed with an average of 12 and 20 probes spanning each gene. This high probe density minimizes signal noise inherent in probe-to-probe sequence-dependent hybridization intensity. We demonstrate that these exon arrays accurately profile genome-wide expression, comparing favorably to currently available arrays and RNA-seq profiling, and can detect alternatively spliced transcript isoforms as well as non-coding RNAs (ncRNAs). Of the 964 candidate alternate splicing events from published RNA-seq studies, 162 are confirmed using the exon array. Furthermore, the exon array predicted 330 previously unidentified alternate splicing events. Gene expression microarrays continue to offer a cost-effective alternative to RNA-seq for the simultaneous monitoring of gene expression and alternative splicing events. Microarrays may even be preferred in some cases due to their affordability and the rapid turn-around of results when hundreds of samples are required for fine-scale systems biology investigations, including the monitoring of the networks of gene co-expression in the emergence of drug resistance.</p></div

Biological replicates show high reproducibility on the exon arrays.

<p>Spearman correlations of biological replicates show highly reproducible signal for 12 hpi samples hybridized to the (A) Nimblegen and (B) Agilent HD exon arrays. (C) Hierarchical clustering of correlation values across all samples hybridized to the high-density exon arrays show reproducibility of signal between chip platforms across the intra-erythrocytic lifecycle.</p

Differentially expressed genes are replicated with high concordance on the exon arrays.

<p>Log₂ ratios of 12 hpi vs. 36 hpi samples were calculated to determine differentially expressed genes. The correlations between log₂ ratios on the Nimblegen and Agilent HD exon (0.905, A and C) and Agilent 15K arrays (0.873, B and D) show that biologically relevant information is replicated across array platforms. The mean differential expression values are comparable between chip platforms, though both Agilent arrays have an overall larger dynamic range (C and D). The correlation between the Agilent 15K and Agilent HD exon array is 0.676 (E). Different biological samples were hybridized to these two arrays and the correlation value matches that of these two biological replicates when hybridized on the same platform (F, 0.692) suggesting that the variation seen is due to differences in the samples.</p

Gene set enrichment analysis (GSEA) of up-regulated genes between exon arrays and previously published 15K agilent array show high reproducibility of biological information.

<p>GSEA plots for chip to chip comparisons of unregulated genes for HB3 12 hpi vs 36 hpi samples. Black lines indicate the location of “hits” from the rank order list of the up or down-regulated genes from the query list. Correlation values between ranked ordered and query list is progressively calculated and indicated by the red bar (with stronger correlations indicated by darker hue). The “zero crosses at” dotted line indicates when the enrichment score decays back to zero and there are no more potential matches between the query and rank ordered lists. (A and C) Comparing the top up-regulated and down-regulated genes on the Agilent 15K array to the rank ordered list of all genes on the Nimblegen exon array. (B and D) Comparing the top up-regulated and down-regulated genes on the Agilent HD exon array to the rank ordered differential expression of all genes on the comparably designed Nimblegen exon array. These comparisons show that the top up-regulated genes between 12 hpi and 36 hpi are consistent across all 3 platforms.</p

Exon array designs.

<p>Exon array designs.</p

Determining the ideal number of probes per gene.

<p>Simulation of the minimum number of probes per gene required to reproduce gene expression levels obtained by using the full probe-set per gene on the exon array. For each time point, gene expression data was obtained by averaging signal intensity across a given subset of probes (2 to 20) and in each case the genome-wide correlation between the expression level obtained when using a subset of probes and the full probe set was determined. To obtain highly correlated signals between replicates, a minimum of 10–12 probes per gene is ideal.</p

Exon arrays can detect exon skipping transcript isoforms.

<p>Comparison of genes with transcript isoforms (A) Differential exon splicing is detected on both Nimblegen and Agilent HD exon array platforms using splicing index with a correlation of 0.626. (B) Venn Diagram showing the overlap of genes with transcript isoforms reported by the Agilent HD Exon Array (blue) and RNA-seq studies (Otto (yellow), Sorber (orange), Broadbent (green), and Lopez-Barragen (purple) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187595#pone.0187595.ref024" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187595#pone.0187595.ref025" target="_blank">25</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187595#pone.0187595.ref036" target="_blank">36</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187595#pone.0187595.ref037" target="_blank">37</a>]), (C) Alternative start sites for PF3D7_0929200 produce time point dependent exon skipping of exons 1 and 2 during the schizont parasite stage at 36 and 48 hpi.</p