The Allen Human Brain Atlas offers three types of searches to allow a user to: (1) obtain gene expression data for specific genes (or probes) of

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1 Microarray Data MICROARRAY DATA Gene Search Boolean Syntax Differential Search Mouse Differential Search Search Results Gene Classification Correlative Search Download Search Results Data Visualization Data Aggregation and Normalization Heat Maps Gene/Probe Detail Multiplanar Viewer For those who are arriving at this database without prior knowledge of genetics or specific genes, a search by disease, pathway, cell type or function may be of most initial use. To do this, click on the relevant search term in the tag cloud. The Allen Human Brain Atlas offers three types of searches to allow a user to: (1) obtain gene expression data for specific genes (or probes) of interest (Gene Search) ; (2) compare expression between different anatomic regions (Differential Search) ; and (3) use a 'seed' gene to find other genes with similar expression patterns (Find Correlates). Gene Search To search for probes associated with a specific gene or group of genes, select the Gene Search radio button and type the gene name, gene symbol, Entrez Gene ID or probe ID in the search box (multiple genes searches require an OR between gene names) before clicking the "Search" button. To show exact matches to your search query click the "Show exact matches only" box.

2 Boolean Syntax The following special operators can be used to build queries: Operator Example Search Type Example Query AND operator: & Gene dopamine & receptor OR operator: Gene ABAT ACHE NOT operator:! Gene "dopamine receptor"!drd3 AND, OR and NOT may be used in place of their corresponding operators. They must be upper case. The AND operator (&) is implicit, so spaces between words that are not separated by an operator will be treated like an &. OR ( ) has higher operator precedence than AND (&). Parentheses can be used to group criteria, but nested parentheses are not supported at this time. The NOT operator (!) is not supported within parentheses. To download your search results, click the "Download this data" link at the bottom left of the heatmap. Differential Search To find genes that show enrichment of expression in one region compared to another region, use the Differential Search mode. Select the "Differential Search" radio button. To find genes or probes with an enhanced gene expression profile in one or more brain regions when compared to one or more other brain regions by entering the target structure(s) in the top search box (separate structures with a semicolon) and your contrast structure(s) in the bottom search box and click the Search button. The search returns will show genes exhibiting higher expression in the target region(s) compared to the contrast region(s).search results are sorted either by p-value or fold change, indicated by the arrow on the buttons over the column of genes. To alter the sort parameter, click on either the "p-value" or the "fold change" button.

3 To filter your results by structure or by donor, click on the "Filter Heatmap" button below the heatmap. To perform the previous search in reverse, click the toggle button to the left of the Search button. To download your search results, click the "Download this data" link at the bottom left of the heatmap. Mouse Differential Search The "Mouse Differential Search" function enables side by side viewing and comparison of differential search across the human microarray and the mouse ISH datasets. Select the "Mouse Differential Search" radio button. To find genes with higher expression in one structure compared to another structure, select a target brain structure in the top drop-down box and a contrast region in the bottom drop-down box and click on the Search button. Search Results The return list is generated by first executing a differential search query in the human microarray dataset. Experiments for each gene are grouped together and ordered by maximum fold-change. For each gene in the human microarray return list, associated experiments in the mouse ISH dataset are identified; fold-change values are computed and displayed. Note that NCBI HomoloGene data is used to link genes across organisms. Click on the dataset column heading to toggle the primary dataset selection. For each experiment, a gestalt visualization of the brain wide expression pattern is also provided. The type of visualization depends on the dataset: For mouse brain data, expression is visualized as rotating 3D thumbnails. Each view is generated by maximum density projection where denser expression area appears more solid and obscures areas of lower density. Each cube is color-coded by average expression intensity ranging from blue (low intensity) through green to red (high intensity). Move slider bar in the header to rotate thumbnail. For human brain data, microarray data is visualized as a heatmap where each column represents a tissue sample in ontological order. Heatmap color represents the z-score over a probe ranging from green (z-score of -3 and below) through black to red (z-score of +3 and above). Hover over any position in the heatmap to obtain the associated tissue sample structural annotation.

4 Gene Classification When you are viewing heatmap data, there will be a "Gene Classification" radio button available. When the gene classification radio button is selected, you can search for enhanced gene expression from one of the categories in the drop down menu. To download your search results, click the "Download this data" link at the bottom left of the heatmap. Correlative Search In using gene expression databases, a "search by example" feature is also highly desirable as genes with similar expression patterns may be related in function. The Find Correlates search utility will accomplish this function. This search by example facility is also available in the Allen Mouse Brain Atlas, Allen Developing Mouse Brain Atlas and the BrainSpan atlas of the developing human brain.

5 Once you have identified a gene of interest, to find other genes with spatial expression profiles similar to your gene of interest, first select your probe by clicking on any cell in the heat map related to that probe. You will see that probe listed in the box above the right hand side of the heat map. Then select the brain structure(s) in which you would like to see a similar expression pattern, and click "Find Correlates". This action will return probes with a similar expression profile to brain region(s) in which you are interested. You can see "anti-correlated" returns by toggling the sort order on column "r" or scrolling to the bottom of the heatmap. You can show less data by turning on the "Filter Heatmap" which will bring up a menu that will allow you to select the donors and structures you'd like to see represented in the heatmap.

6 You can see "anti-correlated" returns by toggling the sort order on column "r" or scrolling to the bottom of the heatmap. To download your search results, click the "Download this data" link at the bottom left of the heatmap. Download Search Results To download your search results, click the "Download this data" link at the bottom left of the heatmap. You will have the option of downloading up to 2000 genes at a time starting from any position in the heatmap. Your start position can be determined from the counter in the top left corner of the heatmap (see screenshot). Your data will be downloaded as three separate files: 1) a Columns.csv file that lists the column headers with associated metadata for each sample, 2) a Probes.csv file that lists the row headers with associated metadata for each probe, and 3) an Expression.csv file that provides a matrix of the expression values for each data point. The column headers, probe headers and expression data will be consistent with the settings from which you downloaded the heatmap (i.e. heatmaps illustrating z-score will result in an Expression.csv file with normalized z-score values).

7 Data Visualization Based on your search, the resulting microarray datasets are presented as a matrix with brain structure (by individual donor) on the horizontal x-axis and gene probes on the vertical y-axis. Because multiple probes were used to measure gene expression for a gene, search returns are reported by probe rather than by gene. The microarray data is presented in a heatmap format where the colors of the heat map correspond either to raw data or to a normalized (z-score) expression level of a probe. Brain structures are organized such that moving left to right on the x-axis is analogous to moving from anterior to posterior first in the cortical areas, followed by subcortical areas, cerebellum and brainstem. In the top right hand corner of the heat map is a toggle button, which when clicked will sort the heat map data either by donor or by structure. Data Aggregation and Normalization Unless viewing data on the lowest possible resolution, the heat maps presented on this site are based on data aggregated within brain structures. That is, when there are multiple samples for a given structure, the value represented in the heat map will be the average of those sample values. We further aggregate the expression values up the ontology tree, eg. Frontal Lobe will have a single averaged expression value, which is the average value for all samples belonging to the frontal lobe. Data represented in the heatmap have been normalized across the entire dataset before they are aggregated, and are normalized again for each probe when the heatmap is constructed. Heat Maps The heatmap is a visualization of the microarray values for the returned probes of interest. Each row of the heat map represents a probe. Each column of the heat map either represents a tissue sample or anatomical brain structure depending on the selected resolution (see below). The colors of the heat map are normalized expression values. Default heat map colors are in the green-red scale where the color green should be interpreted as relatively low expression and red as relatively high expression within the scope of each probe. The color scheme used for the heat map display can be changed to suit the user. You can use the color map bar at the bottom of the heat map to

8 display different normalized (z-score) color representations of the heat map data (i.e. blue-red vs. green-red) or you can visualize log2 normalized expression map where the color scale ranges from dark blue, representing low expression, and passes through cyan, yellow, orange and finally to dark red, representing high expression. The columns of the heat map have different meanings depending on the selected resolution. There are three resolution options available from the drop-down menu under the heat map: Coarse, Structures and Samples. If "Samples" is selected there is a one-to-one correspondence between a column and a physical tissue sample. In the Atlas, there are typically multiple samples for each structure of interest. This oversampling may provide information on variability and spatial gradients. In "Structures" mode, all samples belonging to the same designated structure are combined and averaged together. In "Coarse" mode, the brain is divided into approximately 20 large neuroanatomic divisions or regions (e.g. frontal lobe, occipital lobe, striatum, dorsal thalamus, ventral thalamus). Samples within each partition are averaged together to provide a summary value for the partition. Clicking on a cell of the heat map will bring up detailed information in the area above the heat map. The selected location will be indicated by a black arrow above the heat map. The current location of the cursor will be indicated by the white arrow. In the panel above the heatmap (see image below), information about the sampled anatomical structure is displayed on the left. The stack of structures represents the hierarchy from the structure ontology. Clicking on the link at the bottom of the structure ontology will open up the Interactive Atlas Viewer. For more details on the ontology see the Ontology and Nomenclature whitepaper in the Documentation tab. Additional information is shown in the middle panel, and includes gene symbol, gene name, probe name, the log2 expression value and z-score and links to related data in other Allen Brain Atlas resources. Links to donor meta-data are also included in this panel. Blue text indicates a hyperlink, where there is more information available by clicking on the text. The "Brain Explorer" link, when selected for the first time, will take you to a page where you can download the Brain Explorer 2 3-D viewer software. Once the Brain Explorer program is loaded, clicking on the "Brain Explorer" icon will launch a desktop software application for viewing the Atlas gene expression data in three dimensions. The "Planar View" icon launches the multiplanar viewer that shows the expression profile for the selected probe in the context of the donor brain. Data from multiple brains may be viewed in two ways. By default, columns are grouped first by donors then by structures, allowing a user to compare the full expression profiles of the individual brains side-by-side. Grouping the columns initially by structures then by donors allows a user to view the data for each brain structure with data form all donors grouped side-by-side under that structure. You can toggle between the two groupings by clicking the toggle button over the scroll bar. Comparing Genes of Interest

9 You can add probes of interest to a collection for later viewing. Check the checkbox at the left end of a search result row to add it to your collection. Your choices are stored in a browser 'cookie' on your computer and will remain in effect until you click the "Clear Selections" button, or clear your Web browser's cookie cache. Click the "View Selection Heatmap" button to see all of your selections as a heat map. Alternatively choose View Selection Thumbnails to view your selection as 3-D rotating thumbnails. In Thumbnails mode each row represents a probe with data from each donor arranged from left to right. The color patches in the thumbnail represents a physical tissue sample using the same user-specified color scheme as for heatmap viewing. Clicking on a patch will turn it bright blue and bring up detailed information in the area above the thumbnails. To rotate the thumbnail, drag the black downward triangle on the slider

10 labeled Rotate. You can switch between viewing left cortical, right cortical and sub cortical samples using the Left Cortex, Right Cortex and Subcortex buttons located above the thumbnails. Cortical samples projected on a standard inflated white matter surface while sub-cortical samples are displayed as spheres in their 3D sampling locations. Please note that this feature requires that you have cookies enabled in your browser, which is already the case for the great majority of users. Gene/Probe Detail Under Gene Info, clicking on the Gene Symbol, Gene Name or Probe of your search results will take you to a gene detail page. The microarray gene detail page displays information about the gene (1) including the gene name, aliases, Organism, related data from other Allen Brain Atlas resources, Entrez Gene ID and Chromosome. It also includes Probe metadata (2) including the Probe name, NCBI accession number, sequence length, probe sequence, probe type, transcript GI and GC percentage is also included. Given the discrete nature of the sample collection in these brains, we have visually represented gene expression by structure for each donor and for each probe. You can select probes either by choosing a probe from the drop-down menu or you can click through the pages, each of which outlines a single probe. For each probe, we have provided an anatomical abstract view of the gene expression pattern of each donor. Regions of the brain are listed in each of the views, and the text can be removed by clicking on the 'Labels' button. The cortical gene expression patterns (3) are displayed on an inflated cortical surface (outer and inner surfaces of the left hemisphere). Subcortical structures of the brain are represented from a frontal view (4), and subcortical as well as brainstem structures are shown in a side view (5). Hovering your mouse over an annotated structure will bring up the structure name as well as the normalized and raw expression level values. Clicking on a structure will bring up links to the Multiplanar Viewer and the Brain Explorer. Multiplanar Viewer The Planar View link opens a multiplanar viewer that shows sampling sites for microarray data and indicates gene expression levels for a single probe in coronal, sagittal and horizontal sections of MRI space for a given specimen. Additional viewing frames show spatially-corresponding histological data at the slab and block levels with anatomic annotations and specific delineations of sampling sites for microarray data generation.

11 Multiplanar Navigation This screenshot shows the multiplanar navigation controls. Samples from which microarray tissues were assayed are represented as colored boxes on the MR images and show expression level, according to the active color map. Instead of the MR image, you can select a 3-D thumbnail showing the cortical gene expression. Click or click-and-drag to move the crosshairs on any of the three MRI views ports. The various control and feedback components are labeled here 1-8: Donor: Identification number of the donor currently displayed in the viewer. Probe Name: The label for the probe currently displayed in the viewer. Brain Explorer software Link & Permalink: Click the "Brain Explorer" link to view this gene in the Brain Explorer 3-D viewer. Click the permalink component to generate a URL that will bring you back to this page with the current location settings. Scale, MNI Coordinates & Contrast: Scale bar, MNI coordinates at the crosshairs. Click and drag to move the contrast control. Brain Structure: This field is updated to indicate the structure under the crosshairs. Click on the structure label to launch the ontology browser in a new window. Expression Level: This component reports both the raw expression value and its z-score normalized value. Color Map Window & Level: adjust the upper & lower bounds, or slide the entire control to modify the range of colors used to map expression levels. MRI or Gene Expression Thumbnail View: choose the corresponding MR image or gene expression view from the drop-down menu. Image and Probe Navigation

12 In this screenshot you see the heat map associated with a single, user-defined probe of interest as well as two image viewers illustrating the 2-D histology associated with the point selected by the MRI cross-hairs. The viewer on the left-hand side shows a larger view of the relevant brain slab, with a blue highlight around the block of that slab from which the selected sample was taken. The viewer on the right-hand side shows a detailed view of that block, with structure annotations visible. An arrow on the left hand side of the viewer indicates which direction is dorsal (top of the head). The strip of thumbnail images at the bottom of each viewer shows all of the images from its parent specimen, in sectioning order. Clicking on a thumbnail image in the left-hand viewer will navigate the heatmap and the MRI crosshairs to the current location as well as opening a new set of images in the right-hand viewer. Click on one of the structures to move the MRI crosshairs to the location where that structure was sampled. As you move your mouse over the structures in the right-hand viewer its name will appear in the upper-left corner of the viewer. Navigation tools on each viewer allow you to zoom and pan the individual images using your mouse. The toolbar icons provide more detailed views of the tissue sections: Add or remove annotated structure outline Add or remove annotated structure colors Show all sections from this specimen Show block-face images Launch Single Image Viewer in another window