Fig 1.
Experimental workflow chart depicting generation and organization of raw data and analysis of the transcriptome database.
A microarray analysis was carried out on the brains of T. b. brucei infected mice collected at 0, 7, 14, 21 and 28dpi. Corresponding biotinylated cRNAs were hybridized to the Illumina G6_V2_0_R3_11278593 Chip encoding 45,281 oligonucleotide probes and treated with Streptavidin- generating a normalized 23,213 transcriptionally active probeset. Ten Comparisons distributed between the Infected Series [(0-7dpi)1 (0-14dpi)2 (0-21dpi)3 (0-28dpi)4] and the Temporal Series [(7-14dpi)5 (7-21dpi)6 (7-28dpi)7 (14-21dpi)8 (14-28dpi)9 (21-28dpi)10] generated ten transcriptome datasets which were subjected to three levels of analysis viz i) Differential gene expression analysis, ii) KEGG/GO Functional enrichment analysis and iii) Candidate gene interrogation.
Fig 2.
Volcano Plots with attendant up and down regulated transcript numbers for each Comparison.
Volcano plots that chart statistical significance (p value) versus magnitude of change (fold change) on the y and x axes, respectively was performed on the brains of T. b. brucei infected mice at 0, 7,14, 21 and 28dpi on ten Comparisons comprising the Infected Series (A) and the Temporal Series (B). The number of up-regulated and down-regulated transcripts depicted in red and blue, are aligned below each the ten Comparison and corresponding volcano plot.
Table 1.
Total transcript population plotted against the number of statistically significant mRNAs with ≥ 2 FC reveals variation in the level of gene activity when measured by p value differential gene expression and ≥ 2FC.
Table 2.
Twenty five genes with the highest ± FCs across all Comparisons.
Fig 3.
Representative genes for each of the four temporal generic gene expression patterns.
The majority of differentially expressed genes could be assigned to one of four temporal generic expression patterns over the 28 day timeline designated [7dpi↑] eg (Sfxn3), [7dpi↓] eg Snhg11, [28dpi↑] eg Cxcl9 and [7dpi↑-28dpi↑] eg (|Laptm5). The two 7dpi patterns were by far the most prevalent describing the activity of over 8,000 transcripts between them while [28dpi↑] and [7dpi↑-28dpi↑] chart the activity of over 300 immune genes. In contrast to the representative genes, Sfxn3 and Snhg11 many mRNAs showed more gradation post-14dpi particularly the [7dpi↑] population. Some degree of post-14dpi gradation was also evident than the highly stylized patterns of Cxcl9 and Laptm5.
Table 3.
Quantification of up-and down-regulated KEGG pathways and GO terms for each Comparison.
Fig 4.
KEGG bar graphs for ten selected up and down-regulated KEGG pathways from the (0-7dpi)1 Comparison reveals an early neuronal dominated phenotype.
Ten key up- and down-regulated pathways plotted against p values were selected to represent biological change in the (0-7dpi)1 Comparison.
Fig 5.
KEGG bar graph for ten up-regulated KEGG pathways selected from Comparisons (7-14dpi)5, (7-21dpi)6, (7-28dpi)7 and (0-28dpi)4 depicting an early neuronal dominated phenotype and its transition to an immune-dominated phenotype during disease progression.
Ten key upregulated pathways plotted against p values were selected to represent biological change for each Comparison.
Table 4.
Comparison of cytokine gene expression profiles derived from this study, African trypanosomiasis ID5143 KEGG pathway and the Masocha compilation [3].
Table 5.
Comparison of the chemokine ligand gene expression profiles derived from this study and Masocha compilation [3].