MEPD: Medaka Expression Pattern Database. User manual. 11th September Juan L. Mateo

Transcription

1 MEPD: Medaka Expression Pattern Database User manual 11th September 2015 Juan L. Mateo Centre for Organismal Studies, University of Heidelberg,

2 Contents 1 Introduction 1 2 MEPD homepage 1 3 Browsing data in MEPD Searching gene expression data Searching regulatory element expression data Developmental stages Ontology of anatomical structures Adding and modifying data Gene expression Regulatory element expression Bulk update I

3 List of Figures 1 MEPD homepage. Menu bar highlighted by a red rectangle Options in the search menu. Searching for gene (in situ) or regulatory element expression. 2 3 Options in the Medaka Fish Ontology menu. Ontology of anatomical terms and description of the medaka developmental stages are available here Search form for querying the data on gene expression Searching for gene name Searching for annotated structure Summary result of a search for gene expression Gene details window with link to Ensembl for that gene and to ZFIN.org for the zebrafish orthologous gene Complete result for PAX Filtering by annotated structure Clone details window Search form for querying the data on regulatory elements expression Summary result of a search for regulatory element expression Complete result for a RX2 promoter sequence Construct details window Top of the page showing the description of the medaka developmental stages Tree representing the ontology of anatomical structures Example of searching for the developmental stages in which a specific structure appears Submit menu available for logged-in users Form to add a new gene Form to add a new in situ clone Panel to edit a clone Panel to modify picture properties Panel to select the desired anatomical structure to add as annotation for the current picture Form to add a new construct Panel to edit a construct Spread sheet to upload blulk gene data II

4 1 Introduction This document is designed as a simple user manual to help new users to get acquaintance with MEPD. Here we describe how to take advantage of the application in order to browser the content and also how to add you own data and share it with the rest of the scientific community. First we present the homepage, the next section covers the functionality related to browsing the existing data and, finally, you can find instructions on how to add and modify data in MEPD. 2 MEPD homepage The MEPD homepage (Figure 1) shows a brief description of the database together with two images that represent the two types of expression pattern information that MEPD hosts, namely gene and regulatory element expression. At the top of the page we find the common elements across all the MEPD site, the header and just below the menu bar. This menu bar is highlighted by a red rectangle in Figure 1. Figure 1: MEPD homepage. Menu bar highlighted by a red rectangle. The menu bar allows you to move around the different sections in MEPD. The first button of this menu, Home, will take you to this homepage. Next, there is a submenu, Search, with two buttons to search independently the two kind of expression data. Therefore, you can choose between Search In situ or Search Regulation Data, as shown in Figure 2. 1

5 Figure 2: Options in the search menu. Searching for gene (in situ) or regulatory element expression. Following, there is the Medaka Fish Ontology submenu, which has also two options (Figure 3). The first one, labelled as Anatomy, takes you to a page that shows the ontology defined for the medaka anatomical structures that is used in MEPD. The second button, Developmental Stages, reproduces the description of the hallmarks that define the stages of normal development in medaka as in [1]. Figure 3: Options in the Medaka Fish Ontology menu. Ontology of anatomical terms and description of the medaka developmental stages are available here. After that, there is the Links button, guiding you to a list of useful links related to MEPD and medaka in general. A link to download this manual can be also found in this list. The last button, Login, allows the registered user to validate their username and password in order to enable the functionality to add and modify data. At the bottom of the page we encourage any potential contributor to contact us to add their data. The available link will open a new message in the user s default mail client to send a request to us. 3 Browsing data in MEPD In this section we describe how to get access to the data hosted in MEPD, first for gene expression and later for regulatory elements. 3.1 Searching gene expression data When selecting the button Search In situ from the Search menu, you will see the search form, as shown in Figure 4, to enter the parameters to filter your search. Basically, there are three filter criteria, namely the gene of interest, the anatomical regions annotated and the developmental stages. By default none of these filters are set, so clicking on the button Search will show all available data on gene expression. Figure 4: Search form for querying the data on gene expression. If you want to search only a specific gene you can write the name of that gene or its Ensembl ID in the first text box. The search with this filter is case-insensitive and you don t need to write the full name or 2

6 ID. For instance if you want to query all PAX genes with data in MEPD you can simply write pax as shown in Figure 5. Figure 5: Searching for gene name. When you want to filter by anatomical structures you can start typing and the application will suggest to you the terms from the ontology that are available starting with that characters. An example of this behaviour is shown in Figure 6. Alternatively, you can use the arrow button on the right in which case a drop-down list will appear with all the valid terms. The check box below indicates whether you want to specifically search for the selected structure or, when ticked, if you also want to get gene in which not this very structure is annotated but other that is more specific according to the ontology of anatomical term for medaka. For instance, there might be pictures annotated as with brain expression, and other only with tectum. Obviously, tectum is part of the brain, but if you specify brain in the search form and don t tick the Include Sub-Structures box you will get only the genes with brain annotation. If you would prefer to get all, including tectum, then you have to tick this box. Figure 6: Searching for annotated structure. All the different criteria can be combined, for instance a specific gene with annotated expression in the heart at stage 32. The result of any search will be displayed in a tabular form. For instance, using as query the criteria in Figure 5 the output will be as in Figure 7. This is a summary of the information of all the genes matching the search criteria with the gene name, Ensembl ID and up to three images per gene. In the last column there is a Detail button to get to the full information of the selected gene. It is possible to sort this table by gene name or Ensemble ID clicking on the arrow heads in the column header. The information in this output is grouped by gene. This means that even if there are more than one probe targeting the same gene in the list this gene will only appear once. The Ensembl ID is displayed as a link and clicking on it you can access a new page (Figure 8) from where it is possible to open the Ensembl page of this gene and also, if available, the corresponding orthologous gene in zebrafish with a link to ZFIN.org. The definition of orthologous genes between medaka and zebrafish is taken from Ensembl. 3

7 Figure 7: Summary result of a search for gene expression. Figure 8: Gene details window with link to Ensembl for that gene and to ZFIN.org for the zebrafish orthologous gene. In the case that the number of genes to show is large, like in the default search, the output will be paged. It is possible to select in the search form how many genes per page will be visualized. If the specified query does not match any gene in the database the result will be an empty table. The complete result page for a given gene, accessible through the Detail button, looks like Figure 9 for the case of the gene PAX8. At the top of this table you can find the gene name and the Ensemble ID as a link. This link will take you to the Gene details page (Figure 8) This page displays in the first column the kind of view of the picture showing the expression, typically dorsal, frontal, lateral or detail. The picture itself, which can be visualized in full size clicking on the image. Next, there is the clone name that was used to obtain that result, followed by the stage and the annotated anatomical structures. For the last three columns, if there is more than one value shown in there, below the column name there will be a drop-down list with all the values of that column in the page. The purpose of this list is to filter the output to display only the rows showing the selected value. For instance, if from the PAX8 results from Figure 9 you want only the pictures with annotated expression in the eye you can select this structure in the drop-down list from the colunm Structures. The result, as shown in Figure 10, is that the only one picture with this annotation will be displayed. To go back to the full output you need to select again (all) in the list. From this page it is possible to visualize more information about the clone clicking over it. This information (Figure 11) consists of the clone sequence and its direction, 5 or 3 prime, apart from the target gene name and Ensemble ID. Also clicking on the displayed annotated anatomical structures you can see more information about this annotation, like additional comments or the branch to reach this structure from the ontology. This can be useful to know which structures contain this one annotated in the ontology. 4

8 Figure 9: Complete result for PAX8. Figure 10: Filtering by annotated structure. 3.2 Searching regulatory element expression data Searching for regulatory elements expression patterns is very similar to the search for gene expression patterns. In fact the search form is identical to the previous one (Figure 12). The only difference is that, obviously, when filtering for gene this does not refer to the gene one wants to query but the gene that is associate to the regulatory element being the closest in the host genome either up- or down-stream. Now the result of the search is again similar to the previous case but there will be two columns referring to genes, up and down-stream. In addition, this table is indexed by construct, therefore there can be two or more rows for different constructs but associated to the same genes. Apart from these differences, the tabular output has the same behaviour as we showed before for gene expression. An example of this output is in Figure 13. The detail output for regulatory elements is, again, giving the same functionality as for gene expression. In Figure 14 you can see the complete result for a promoter of the RX2 gene. For constructs there is more information than for in situ clones. Figure 15 shows the information for the 5

9 Figure 11: Clone details window. Figure 12: Search form for querying the data on regulatory elements expression. construct hosting the RX2 promoter. In this case, apart from the sequence responsible of the expression pattern, there is also information about the coordinates and species of origin. This is necessary since it is possible to analyse regulatory sequences from other species in medaka. It is also possible to see how the construct was generated with the base cloning vector, the reporter gene, the promoter used, if applicable, and the insertion type. 3.3 Developmental stages In this page (Figure 16) you can check the description of the developmental stages in medaka as defined in [1]. This content allows you to identify easily certain properties in the developing embryo to stage it consistently with the rest of data in MEPD. 3.4 Ontology of anatomical structures This section of MEPD display to you the ontology of anatomical structures used to annotate the expression patterns. Apart from defining a controlled vocabulary of terms, this ontology also specifies a hierarchy between them. This serves two purposes, first, it is easier to navigate through the terms, either top down or down top, from more general to more specific structures. Second, this makes possible to specify a query search containing substructures, as we have describe earlier. To get the most benefit of it, you should be familiar with this ontology and this is the purpose of this page. As shown in Figure 17, the ontology is defined for each developmental stage separately. You can open or 6

10 Figure 13: Summary result of a search for regulatory element expression. Figure 14: Complete result for a RX2 promoter sequence. close each tree by clicking on the on the + or - boxes respectively. In this way you can explore the full tree and identify the terms that you are looking for. In some cases it will be convenient to know in which stages a specific term is present. For that you can use the search form at the top of the page. From the drop-down list you can select one of the terms in the ontology and after clicking on the Search button to the right of the ontology tree it will appear in red the list of stages where you can find it. This behaviour is exemplified in Figure Adding and modifying data MEPD is not only the private database of a single group, but aims at being a common repository of the whole medaka community for expression patterns. To achieve this and allow that everyone can easily share their data we have taken care to provide an easy and simple way to add new information to MEPD and keep it up-to-date. Adding or modifying data in MEPD is only possible for registered user, however getting an account is as simple as requesting that by . Using the Login button in the menu you can introduce your credentials to be validated. In case of a successful login the menu bar will change substituting the Login button by Logout to close the session and adding a new submenu, Submit Data. 7

11 Figure 15: Construct details window. In this submenu (Figure 19) the first button is used to add new genes that are not yet included in MEPD. Below that there are two additional submenus to add or modify gene or regulatory element expression data respectively. To add a new gene (20) you only need to enter the corresponding gene name and Ensembl ID. Since from the result pages all genes are linked to the Ensembl web site, additional information is accessible and more up-to-data from this web site. However, there is also available a box to state comments. This can be used to specify additional information, specially important if you need to add a gene that is not yet annotated in Ensembl. 4.1 Gene expression Adding new gene expression data is done in two steps. First it is necessary to include the information about the clone in the database if it is not yet there. The form to do so is shown in Figure 21 and in there you can specify the target gene, a name for the clone, the sequence and the direction in which it is given, 5 or 3 prime. The target gene can be given either by the gene name or by the Ensembl ID, automatically the other field will be filled. In both fields the system will show the genes that match with the text entered to help specifying a valid value. Once the clone is included in MEPD the next step to add additional information is using the Manage clones button form the menu. In this case you will see a table with all the clones available in MEPD. From this list you need to select the clone that you want to modify or add new information. You can search by gene name, Ensembl ID or clone name just typing the values to filter each column in the text field below the column header. In Figure 22 we show how to search for clones targeting the PAX8 gene. In this case there are two clones for this gene. After you click over the corresponding row, in the right side of the table you will see a summary of information for the clone with the pictures associated with it. Some of the button may be disable, like Remove in this example, depending of the user rights. 8

12 Figure 16: Top of the page showing the description of the medaka developmental stages. To add a new picture simply click on the Upload picture button. A new panel will be visible with the Picure properties. This panel will be similar to the one shown in Figure 23 but values and an Add button instead of the picture. Click on this Add button to upload an image from you computer to the database. Once the upload is successful the picture will be visualized and you can continue specifying the rest of the fields. Specially important is to indicate properly the stage of the embryo in the image because only the terms from the ontology consistent with that stage can be added. Now, to add new annotation to the picture, i.e. to specify which anatomical structure are stained, you can click on the New structure button. Then you will see a new panel (Figure 24) with the ontology corresponding to the specified stage. From this panel you can select the structure you want to assign by clicking on the name. 4.2 Regulatory element expression As for the search functionality, adding and modifying information for regulatory elements follows the same principle and flow. In Figure 25 you can see the form to add a new construct. It has the same structure as for in situ clones but only with different fields. The same is true for editing the information about construct as shown in Figure 26. In this case we filter the list of constructs by the construct name. 4.3 Bulk update The online forms to modify data in MEPD are most suitable to add small amount of clones or constructs and to add and correct annotations. However, for large amount of data, for instance in case of small to large screening experiments, a bulk update is more convenient. 9

13 Figure 17: Tree representing the ontology of anatomical structures. To make a bulk update you only need to fill a spread sheet with the information to add to the database. You can find a template of this spread sheet in the Links page, there is one for genes and other for regulatory elements data. Once the spread sheet is filled with the information you want to add you can send it to us together with the corresponding pictures. In these templates the columns are labelled indicating to which field in the database they correspond, and coloured by sections, clone, sequence, picture and annotation. In Figure 27 we show an example of the template for gene expression data filled with two rows. In this case we would like to add a clone with one picture and to annotate two structures. For that we replicate in both rows the information about clone and picture. This can be easily done using the Copy & Paste function and assures that the different pictures and annotations are all linked to the proper clone. This means that you should provide as many rows with the same content for clone and picture as annotations for that picture you want to add, and in total as many rows with the same content for clone as the number of pictures and annotations for each one. References [1] Iwamatsu T Stages of normal development in the medaka Oryzias latipes. Mech Dev 121:

14 Figure 18: Example of searching for the developmental stages in which a specific structure appears. Figure 19: Submit menu available for logged-in users. Figure 20: Form to add a new gene. Figure 21: Form to add a new in situ clone. 11

15 Figure 22: Panel to edit a clone. Figure 23: Panel to modify picture properties. Figure 24: Panel to select the desired anatomical structure to add as annotation for the current picture. 12

16 Figure 25: Form to add a new construct. Figure 26: Panel to edit a construct. Figure 27: Spread sheet to upload blulk gene data. 13