Introduction

An IGB user contacted us with a request to add a new genome to the IGB QuickLoad system - the Xenopus tropicalis genome assembly dated November, 2009, also called JGI 4.2/xenTro3. (See: http://genome.ucsc.edu/cgi-bin/hgGateway?hgsid=262072157&clade=vertebrate&org=X.+tropicalis&db=0).

Because this genome is supported at the UCSC Genome Browser, adding it to the system should be fairly straightforward. All we need to do is download the data, format it, and move it onto the main IGB QuickLoad site currently hosted at UNC Charlotte, in the Bioinformatics and Genomics Department's server room.

Methods

Downloading sequence data (2bit file) from UCSC Genome Bioinformatics

Step one is to download the sequence data.

The UCSC Web site provides this page that lists the different organisms supported in their system. I followed the link to the X. tropicalis genome and clicked the link labeled Full data set, which took me to the "bigZips" page here: http://hgdownload.cse.ucsc.edu/goldenPath/xenTro3/bigZips/.

Fortunately, it appears that UCSC already provides the fasta sequence data in 2bit format, saving me a step.

First, I added a new directory for this genome to my checked-out copy of the IGB QuickLoad subversion repository, like so:

Next, I changed in the new directory and then downloaded this 2bit file using wget in the usual way:

Once the file downloaded, I changed the name and checked the file size:

I changed the name because IGB expects the genome sequence file name to match the genome version name. That is, when a user requests the sequence to be loaded into IGB, IGB will look for a file named X_tropicalis_Nov_2009.2bit and then use that file to retrieve the sequence data.

I checked the file size to determine if it would be reasonable for me to add the file to repository. I've done this for all the plant genomes we support, mainly as a convenience for mirroring the QuickLoad site on different locations. If the sequence file is version controlled, I can easily check it out to a new location and also associated notes via my commit messages. However, if the file were prohibitively large (> 1000 megabytes, for example), then I might use another strategy to deploy the file. But this size (373M) isn't too bad, so I'll add it to the QuickLoad subversion repository in the usual way:

Creating genome.txt using twoBitInfo

Next, I used twoBitInfo to make a "genome.txt" file reporting the names of the assembled chromosomes and contigs and their sizes:

This creates the genome.txt file IGB needs to display contig and chromosome names and their sizes in the Current Genome tab.

Curious about how complete the assembly is, I counted the contigs:

Yikes! That's a lot. Probably the genome is not quite close to being finished. But I know from experience with other less complete genomes that IGB should be able to handle such a large number of contigs. Users who want to view the largest ones can sort on size in the Current Genome table, which displays the contigs and their sizes.

So I added to the repository and committed it like so:

Downloading genome annotations (ESTs and RefSeq genes) from UCSC Table Browser

My plan (currently) is to get the RefSeq genes track from UCSC and deploy it on our QuickLoad site. I'll provide meta-data about the data set using the annots.xml file. (More on this later.)

Next, I used the UCSC Table Browser to get the RefSeq genes for this species. Here are the settings I used for this:

Unfortunately, there don't appear to be a lot of RefSeq gene annotations available for this species:

Probably it would be a good idea to look for another data set that might provide a more complete view of the Xenopus expressed gene repertoire.

Using the Table Browser, I explored the available data sets for Xenopus. To do this, I just choose a table and then click the button "describe table schema," which leads to a page reporting the number of annotations available in the selected table.

It looks like the table all_mrna may be the most complete; it contains slightly more than 20,000 rows. So, probably users will want to see this data, as well as the RefGene track. I'll download this data set, add it to the repository, and add it to the annots.xml file (see below.)

I also checked the ESTs data set. There are around 1.5 million ESTs for Xenopus. I'll download that data set as well, but set it up so that users can access it on a region-by-region basis by sorting and indexing using the tabix utility. I'll also need to massage the format a bit to get it to make the PSL (blat output) format.

Processing ESTs using tabix to support fast access in IGB

To process the EST data set, I used the following command to strip off the first column

Next, I sorted and created an index using bgzip and  tabix:

The sort command first sorts on fields 14 through 14, inclusive (-k 14,14) and then sorts on field 16 through 16, inclusive (-k16,16). The first sort (field 14) sorts the file by target sequence name and the second sort (field 16) sorts numerically on the start position for each alignment. After sorting, bgzip block-compresses the file. 

The last command (tabix) creates a tabix index (.tbi) file that IGB knows to look for when it encounters files with extension .gz.

Next, I added each of the data files to IGB QuickLoad subversion repository - the sorted, bgzip-compressed EST data file, its index file, a compressed (gzip) file for the RefGene data set, and a compressed, gzip'd file for the mRNA data set. Since both will be loaded as soon as the user visits the genome version, I did not bother to make tabix'd versions of those files.

Deploying the files on QuickLoad

I've created a number of data files, and so my next step will be to try opening them in IGB. I also want to test whether IGB will be able to open and display the genome sequence.

So my next step is to create an annots.xml file IGB will use to get a listing of the annotations available for this genome as well as styling information, e.g., the background colors to use for gene annotations, whether to load all the annotations immediately, and so on.

Creating new annots.xml file for Xenopus genome and annotations

Being a bit lazy, I usually just copy and paste another annots.xml file from another part of the repository when setting up a new genome. I use svn for this, however:

I then open a simple text editor (like TextEdit) and edited the file, like so:

Recently, we added the capability to specify various styles for annotation files delivered via QuickLoad. Most of these (e.g., foreground and background) are self-explanatory, but a few are not so obvious as they pertain to specialized aspects of how IGB presents data.

The "max_depth" parameter refers to the maximum number of overlapping annotations that can appear in a stack within a track. The "name_size" parameter specifies the font size for the track labels that appear on the left-hand side of the main display menu. The "url" parameter specifies where IGB links to when the user clicks the info button (blue "i" icon) next to a data set. In this case, we link back to the main UCSC Web page, since this is where the data came from originally. The "name" parameter indicates the file name IGB should load, and the "description" parameter specifies the tooltip text that will appear when the user hovers the mouse over the data set in the Data Access Panel. The "title" specifies the name of the data set as it will appear to the user.

Testing new genome version directory in IGB

Finally, I added my local QuickLoad site to IGB (using the Configure link in the Data Access panel) and loaded up the new genome.

Everything looked good, and after making a few changes to the color scheme (to maximize legibility), I committed my final changes to the repository.

My last step was to log into the main IGB QuickLoad site and run "svn up" to deploy the new genome and all its associated data files on the main site to ensure that all IGB users can now access the data.

Conclusion

All in all, the entire process, subtracted breaks for lunch and meetings, took about four hours, including the time it took to write this tutorial.

Visualization of the newly added genome

Here is what the genome will look like for some-one visiting this genome version for the first time: