Assembly Hubs

Overview

The Assembly Hub function allows you to display your novel genome sequence using the UCSC Genome Browser.

Web Server

To display your novel genome sequence, use a web server at your institution (or GBiB) to supply your files to the UCSC Genome Browser (please note, hosting hub files on HTTP is highly recommended and much more efficient than FTP). You then establish a hierarchy of directories and files to host your novel genome sequence. For example:

myHub/ - directory to organize your files on this hub
     hub.txt – primary reference text file to define the hub, refers to:
     genomes.txt – definitions for each genome assembly on this hub
          newOrg1/ - directory of files for this specific genome assembly
               newOrg1.2bit – ‘2bit’ file constructed from your fasta sequence
               description.html – information about this assembly for users
               trackDb.txt – definitions for tracks on this genome assembly
               groups.txt – definitions for track groups on this assembly
               bigWig and bigBed files – data for tracks on this assembly
               external track hub data tracks can be displayed on this assembly

The URL to reference this hub would be: http://yourLab.yourInstitution.edu/myHub/hub.txt

You can view a working example hierarchy of files at: Plants
A smaller slice of this hub is represented in a Quick Start Guide to Assembly Hubs.

hub.txt

The initial file hub.txt is the primary URL reference for your assembly hub. The format of the file:

hub hubName
shortLabel genome
longLabel Comment describing this hub contents
genomesFile genomes.txt
email contactEmail@institution.edu
descriptionUrl aboutHub.html

The shortLabel is the name that will appear in the genome pull-down menu at the UCSC gateway page. Example: Plants

The genomesFile is a reference to the next definition file in this chain that will describe the assemblies and tracks available at this hub. Typically genomes.txt is at the same directory level as this hub.txt, however it can also be a relative path reference to a different directory level.

The email address provides users a contact point for queries related to this assembly hub.

The descriptionUrl provides a relative path or URL link to a webpage describing the overall hub.

genomes.txt

The genomes.txt file provides the references to the genome assemblies and tracks available at this assembly hub. The example file indicates the typical contents:

genome ricCom1
trackDb ricCom1/trackDb.txt
groups ricCom1/groups.txt
description July 2011 Castor bean
twoBitPath ricCom1/ricCom1.2bit
organism Ricinus communis
defaultPos EQ973772:1000000-2000000
orderKey 4800
scientificName Ricinus communis
htmlPath ricCom1/description.html

There can be multiple assembly definitions in this single file. Separate these stanzas with blank lines. The references to other files are relative path references. In this example there is a sub-directory here called ricCom1 which contains the files for this specific assembly.

• The genome name is the equivalent to the UCSC database name. The genome browser displays this database name in title pages in the genome browser.
• The trackDb refers to a file which defines the tracks to place on this genome assembly. The format of this file is described in the Track Hub help reference documentation.
• The groups refers to a file which defines the track groups on this genome browser. Track groups are the sections of related tracks grouped together under the primary genome browser graphics display image.
• The description will be displayed for user information on the gateway page and most title pages of this genome assembly browser. It is the name displayed in the assembly pull-down menu on the browser gateway page.
• The twoBitPath refers to the .2bit file containing the sequence for this assembly. Typically this file is constructed from the original fasta files for the sequence using the kent program faToTwoBit. This line can also point to a URL, for example, if you are duplicating an existing Assembly Hub, you can use the original hub's 2bit file's URL location here.
• The organism string is displayed along with the description on most title pages in the genome browser. Adjust your names in organism and description until they are appropriate. This example is very close to what the genome browser normally displays. This organism name is the name that appears in the genome pull-down menu on the browser gateway page.
• The defaultPos specifies the default position the genome browser will open when a user first views this assembly. This is usually selected to highlight a popular gene or region of interest in the genome assembly.
• The orderKey is used with other genome definitions at this hub to order the pull-down menu ordering the genome pull-down menu.
• The htmlPath refers to an html file that is used on the gateway page to display information about the assembly.

Note that it is strongly encouraged to give each of your genomes stanza's a line for defaultPos, scientificName, organism, description (along with other above settings) so that when your hub is attached it will load a specified default location and have text to be more easily searched from the Gateway page.

2bit file

The .2bit file is constructed from the fasta sequence for the assembly. The kent source program faToTwoBit is used to construct this file. Download the progrem from the downloads section of the Browser. For example:

faToTwoBit ricCom1.fa ricCom1.2bit

Use the twoBitInfo to verify the sequences in this assembly and create a chrom.sizes file which is not used in the hub, but is useful in later processing to construct the big* files:

twoBitInfo ricCom1.2bit stdout | sort -k2rn > ricCom1.chrom.sizes

The .2bit commands can function with the .2bit file at a URL:

twoBitInfo -udcDir=. http://genome-test.cse.ucsc.edu/~hiram/hubs/Plants/ricCom1/ricCom1.2bit stdout | sort -k2nr > ricCom1.chrom.sizes

Sequence can be extracted from the .2bit file with the twoBitToFa command, for example:

twoBitToFa -seq=chrCp -udcDir=. http://genome-test.cse.ucsc.edu/~hiram/hubs/Plants/ricCom1/ricCom1.2bit stdout > ricCom1.chrCp.fa

groups.txt

The groups.txt file defines the grouping of track controls under the primary genome browser image display. The example referenced here has the usual definitions as found in the UCSC Genome Browser.

Each group is defined, for example the Mapping group:

name map
label Mapping
priority 2
defaultIsClosed 0

• The name is used in the trackDb.txt track definition group, to assign a particular track to this group.
• The label is displayed on the genome browser as the title of this group of track controls
• The priority orders this track group with the other track groups
• The defaultIsClosed determines if this track group is expanded or closed by default. Values to use are 0 or 1

Building Tracks

Tracks are defined in the trackDb.txt where each stanza describes how tracks are displayed (shortLabel/longLabel/color/visibility) and other information such as what group the track should belong to (referencing the groups.txt) and if any additional html should display when one clicks into the track or a track item:

track gap_
longLabel Gap
shortLabel Gap
priority 11
visibility dense
color 0,0,0
bigDataUrl bbi/ricCom1.gap.bb
type bigBed 4
group map
html ../trackDescriptions/gap

For more informations about the syntax of the trackDb.txt file, use UCSC's Hub Track Database Definition page

It helps to have a cluster super computer to process the genomes to construct tracks. It can be done for small genomes on single computers that have multiple cores. The process for each track is unique. Please note the continuing document: Browser Track Construction for a discussion of constructing tracks for your assembly hub.

Adding BLAT servers

By running your own blat server with gfServer you can add lines to the genomes.txt file of your assembly hub to enable the browser to access the server and activate blat searches.

• First run two instances of gfServer from http://yourLab.yourInstitution.edu at the location of yourAssembly.2bit file, specifying a port that the gfServer will be accessible from for amino acid (-trans option) and DNA searches. Please note the -mask option will ignore all lower-case assembly sequence, which is the convention the UCSC Browser uses for masked sequence, so you may not want to include it from the example below.

Selecting a port:

When picking a port number, stick with numbers between 1024 and 49151. Anything less than 1024 is considered a system port and you'll need to be root in order to open it. Anything above 49151 is considered dynamic and randomly assigned. If you're starting a server that you will use a web browser to connect to, it is suggested to choose something with 8's in it, since that's the tradition. 8080, 8000, and 8888 are all popular, but other open ports will work just fine.

For example, these two lines will specify port 17777 for amino acid searches and 17779 for DNA searches and are run from the publicly accessibly directory location of yourAssembly.2bit file:

gfServer start localhost 17777 -trans -mask yourAssembly.2bit &
gfServer start localhost 17779 -stepSize=5 yourAssembly.2bit &

• Next edit your genomes.txt stanza that references yourAssembly to have two lines to inform the browser of where the blat servers are located and what ports to use. See an example of commented out lines here. Please note the capital "B" in transBlat.

transBlat yourLab.yourInstitution.edu 17777
blat yourLab.yourInstitution.edu 17779

• You should now be able to load and perform blat operations on your assembly. For example a URL such as the following would bring up the blat CGI and have your assembly listed at the bottom of the "Genome:" drop-down menu: http://genome.ucsc.edu/cgi-bin/hgBlat?hubUrl=http://yourLab.yourInstitution.edu/myHub/hub.txt

• Some institutions have firewalls that will prevent the browser from sending multiple inquiries to your blat servers, in which case you may need to request your admins add this IP range as exceptions that are not limited: 128.114.119.* That will cover the U.S. genome.ucsc.edu site. In case you may wish the requests to work from our European Mirror genome-euro.ucsc.edu site, you would want to include 129.70.40.120 also to the exception list.

Please see more about configuring your blat gfServer here to replicate the UCSC Browser's settings. The Source Downloads page offers access to utilities with pre-compiled binaries such as gfserver found in a blat/ directory for your machine type here and further blat documentation here, and the gfServer usage statement for further options.

Please also know you can set up gfservers on a GBiB and run it locally. Please see this GBiB assembly blat step-by-step set up page for details.

Note: You can stop your instance of gfServer with a command. For example:

gfServer stop localhost 17860 

Troubleshooting BLAT servers for your hub

The following is an example of an error message
when attempting to run a DNA sequence query
via the web-based BLAT tool after loading a hub,
after starting a gfServer instance (from the same dir as the .2bit file).
For example, a command to start an instance of gfServer:

$ gfServer start localhost 17779 -stepSize=5 contigsRenamed.2bit &

Example of a possible error message, from web-based BLAT
after attempting a web-based BLAT query:

Error in TCP non-blocking connect() 111 - Connection refused
Operation now in progress
Sorry, the BLAT/iPCR server seems to be down. Please try again later.

Check the following:

Process check

First, make sure your gfServer instance is running.
Type the following command to check for your running gfServer process:

$ ps aux | grep gfServer

Check for correct path/filename

In your genomes.txt file, does your twoBitPath/filename match what you specified in your command to start gfServer?

In your genomes.txt file, is the location of the instance to your gfServer correct?

To check this, you can cd into the directory where you started your gfServer, then type the command:

$ hostname -i

Your result should be an IP address, for example, "132.249.245.79".

Now you can test the connection to your port that you specified, with a simple telnet command.

Type in the following command: telnet yourIP yourPort. For example:

$ telnet 132.249.245.79 17777

The results should read, "Connected to 132.249.245.79".

Otherwise, if gfServer isn't running or if you typed the wrong location in your telnet command, telnet will say, "Connection refused."

In this example, check your genomes.txt file, and make sure your blat line reads, "blat 132.249.245.79 17777".

You may need to change your genomes.txt file from, for example, "blat localhost 17777" to "blat 132.249.245.79 17777" (use your specific IP/host name where gfServer is running).

Check "gfServer status" check

In the directory of your .2bit flle (should be the same dir where you started gfServer), type: gfServer status yourLocation yourPort .
For example:

$ gfServer status 132.249.245.79 17777

You should see output like this:

version 36x2
type nucleotide
host localhost
port 17777
tileSize 11
stepSize 5
minMatch 2
pcr requests 0
blat requests 0
bases 0
misses 0
noSig 1
trimmed 0
warnings 0

Testing with gfClient

The best troubleshooting test is to take the webpage out of the equation, and use the command line utility, gfClient, to run the query on your instance of gfServer. If you can successfully connect gfClient to gfServer, you will know that your location and port specification are correct.

From the directory that holds your hub's .2bit file (should be the same directory where your instance of gfServer was launched), perform a query using gfClient:

You can type "gfClient" on your command line to see the usage statement.

Use the following command: gfClient yourLocation yourPort pathOf2bitFile yourFastaQuery.fa nameOfOutputFile.psl

For example:

$ gfClient localhost 17777 . query.fa gfOutput.psl

Note the " . " after the port, to specify that the query will use the .2bit file in the current directory.

After running this command, take a look at the gfOutput.psl file. If successful, you will see BLAT results.