Additional file 1 of Global assessment of organ specific basal gene expression over a diurnal cycle with analyses of gene copies exhibiting cyclic expression patterns

Additional file 1: Supplement Figure 1. Sample collection and experimental setup: 9 organs (skin, brain, liver, gill, heart, muscle, testis, ovary and eye) were collected at 5 am, 8 am, 11 am, 2 pm, 5 pm, 8 pm, 11 pm and 2 am. Duplicate samples of total RNA from each organ of individual fish was isolated for gene expression profiling. Supplement Figure 2. Flowchart depicting the configuration of the gene expression browser. Establishment of the gene expression browser includes 2 main steps: 1. Sample collection and gene expression profiling, and Gene ID and Gene name conversion: Gene expression data were normalized and scaled and stored in the XGSC website; All Xiphophorus Ensembl gene ID and corresponding gene name, as well as other bioinformatics information were downloaded into a custom table and stored in the XGSC website; 2. Configuration of the user interface and server: user interface was designed to convert common gene name to Ensembl gene ID to precisely identify the gene of interest, and to show basal gene expression pattern. These tools are stored in both shiny.io and Github for user to launch the browser remotely or locally. Supplement Figure 3. Illustration of the gene ID converter. The user interface (UI) contains a gene ID conversion tool. Users can input a common gene name as a key word to search through the whole genome to identify genes that may be of study interest. An example of “per” for period gene family is used in the Fig. 9 lines of data were returned. Among those two are gper1, a GPCR gene, and three are perp, a TP53 apoptosis effector gene that are all not relevant to the per gene family, and 4 per gene family members: per1b, per2, per3 and a per2 paralog (ENSMAG00000006651). Supplement Figure 4. Illustration of the gene expression browser user interface. Once in the web tool at the Xiphophorus Genetic Stock Center web page, an outside user can enter a proper Ensembl gene ID as input in order to generate a bar graph representing the organ-specific average expression levels of the query gene over a 24-h period. This generates an organ-specific expression curve depicting the gene expression at each time point of a day. The example shown is per1b. The table underneath the bar graph and expression pattern plot shows basic statics of whether this gene exhibits a cyclic expression pattern, including gene name, description, peak time (in Zt), peak shape (i.e., hours to go from peak expression to the lowest expression level), amplitude (i.e., distance between peak expression and the bottom of a circadian expression pattern). Supplement Figure 5. Cyclic genes and opsin expression heatmaps representing (a) circadian genes distribution in each organ and (b) opsin expression in each organ. For each heatmap, each row represents a gene and each column represents an organ. Yellow blocks show a gene (a) exhibiting circadian expression pattern, or (b) expressed. Supplement Figure 6. Circadian master regulator gene orthology to gar. Three pairs of circadian master regulators in Xiphophorus were found to be Ohnologs. These genes include (a) arntl2, (b) per2, (c) cry1ab. A syntenic block plot is used to show shared synteny of each Ohnolog with a gar ortholog. Red dots (open or closed) represent upstream and downstream neighboring genes of a Xiphophorus gene. The larger closed red dot represents a particular circadian gene. Blue dots (open or closed) represent upstream and downstream neighboring genes of gar ortholog of the Xiphophorus gene, and the larger closed blue dot represents the gar ortholog. Each Xiphophorus gene in the chromosome window is used to scan the gene of gar ortholog neighboring genes for orthology. If orthology exists, the Xiphophorus gene and the corresponding gar gene are connected by a gray line, with a closed dot representing Xiphophorus gene. Supplement Figure 7. Gene expression patterns of cyclic gene Ohnologs. A total of 626 Ohnologs pairs exhibited at least one of the duplicate showing circadian expression cycle in one organ. Per Ohnolog pair, 9 plots representing 9 organs were represent basal gene expression patterns, with solid line and dashed lines represented each gene duplicate. Supplement Figure 8. Gene expression patterns of singletons. A total of 4573 singletons exhibited circadian expression cycle in at least one organ. Per gene, 9 plots representing 9 organs were represent basal gene expression patterns, with solid line and dashed lines represented each gene duplicate. Supplement Figure 9. Expression analyses of cyclic gene Ohnologs exhibiting functional divergence in expression pattern: (A) intra-organ functional divergence, and (B) inter-organ functional divergence. In each case, two heatmap panels were used to describe (left panel) the expression status (i.e., gray = not expressed, black = expressed) and (right panel) relative expression between the Ohnologs (Ohnolog1 expression / Ohnolog2 expression). For the left panel, the center red line dividing each panel, splits the left and right half that represent Ohnolog1 and Ohnolog2, respectively. Status of expression and relative expression of Ohnologs are not correlated to if one Ohnolog exhibit circadian pattern, but not the other. S=Skin; B=Brain; O=Ovary; H=Heart; M = Muscle; E = Eye; G = Gills; L = Liver; T = Testis. Supplement Figure 10. Validating expression patterns of 4 cyclic genes using nanostring nCounter. Expression patterns of 4 cyclic genes identified in X. maculatus skin using RNA-Seq (arntl1, cry1ab, clockb, clocka) were assessed using nCounter by direct transcript molecule counting.