Technical document. Section 1 Using the website to investigate a specific B. rapa BAC.

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1 Technical document The purpose of this document is to help navigate through the major features of this website and act as a basic training manual to enable you to interpret and use the resources and tools involved the annotation of the B. rapa BACs. It will also cover using other resources found on this website such as the Brassica 95K microarray database and the local blast server. These issues will be addressed by running through a real example. Section 1 Using the website to investigate a specific B. rapa BAC. There are 3 different ways you can search for a BAC on the website, these are, 1. Using the link to display all the BACs. This will instantiate a pop-up window that displays all the BACs that are currently available in the database. From there, you can scroll through the list and click on the name of the BAC of your choice, which will in turn, direct you to the Gbrowse annotation display for that clone. 2. Using the link to display the most recently annotated BACs. Again this works in the same way as the above, only this link will display the BACs that have been added to the database in the last 31 days.

2 3. Using the BAC name search search box. In this box you can type the name of the BAC. The term you enter is then used to search the database and come up with any results that match your entry in the form of a pop-up window, from there you can click on the BAC of your choice and that will take you to the annotation page of that BAC. The search is case insensitive and wild cards are enabled, so, for example, if you wanted to to retrieve all KBrH BACs, then you can simply type that in the search box and all the KBrH library BACs will be displayed. Type KBrB123I09 in the search box and follow the link from the pop-up window to the annotation page for that BAC by clicking on the BAC name itself.

3 This will display the annotation page for this BAC. By default, the tracks that are enabled are, a. The PASA gene model alignments. b. The A. thaliana gene model alignments. c. Brassica EST transcript assembly alignments.

4 You can use your mouse to hover over one of the PASA gene models, this will initiate a tooltip displaying information about that gene model and a link ( show virtual protein sequence ) of that gene model. Clicking on this link will initiate another display that displays information about the individual CDS features of the predicted gene. At the top of this display, you can find a button Start Jalview, which will instantiate a jalview alignment pop-up display. You can use the jalview display to scroll through the alignment of the gene model and visually inspect it. If you scroll down to the bottom of the main Gbrowse annotation display of the BAC, you can see all the different annotation tracks that are available, these are grouped into 6 major categories, they are, a. Overview. b. Analysis. c. Gene Predictions. d. General. e. Markers. f. Transcript assemblies. You can select to display any of the tracks that you are interested in by checking the corresponding tick box and clicking on Update image.

5 Clicking on the title of the track will open a new page that displays more detailed information describing what that track actually is. There are various different ways in which you can manipulate the display of the BAC annotation, but this document will not be covering them, a more detailed tutorial on how to use the Gbrowse display can be found here. The Landmark or Region search box in the Gbrowse annotation page can be used to extensively search the BAC database. You can use it to search for BACs, Arabidopsis gene models that are aligned to the rapa BACs, EST transcripts etc... Essentially, you can use this search box to search for any information about the different tracks or the BACs, or even by using keyword searches. Have a look at the examples section in the annotation page.

6 The sections that were explained above cover a main walk-through of how to use the site to investigate B. rapa BAC annotations. You should now navigate back to the homepage of the project to continue with this tutorial. Section 2 local Blast server and Blastable databases. We will now look at the local blast server hosted on this site. You can find this local blast implementation by following the Brassica Blast Server link on the main page. This will initiate a pop-up window, which can be used to blast your sequence against the following databases, 1. Sequenced KBr BACs. 2. JBnB (B. napus) BACend sequences. 3. KBr (B. rapa) BACend sequences. 4. All BrassicaDB sequences 5. EST assemblies (95K unigenes) 6. BrassicaDB-protein. 7. Arabidopsis pseudochromosomes. 8. Arabidopsis genes. 9. B. oleracea GSSs.

7 A blast search is also implemented explicitly on the Brassica 95K unigene set and this will blast your sequence against all the unigenes that went into implementing the Brassica 95K microarray (more information about the Brassica microarray can be found at????). Following the blast vs unigenes link will display a pop-up window of the blast submission form similar to the previous example. There is also a database, which holds information about the unigenes that went into the making of the microarray and a guide on how to interrogate the database. This guide is available by following the Read me link in the Brassica 95K unigene section of the main page. An ftp site is also available where various data files relating to the project can be found. You can also look at the README file on the ftp site to understand what information all the different files on the ftp site contain. The link to the ftp site is available on the main homepage.