Difference between revisions of "ABRF2010 Workshop"
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User: your profile name from the morning session
User: your profile name from the morning session
Revision as of 20:44, 20 March 2010
GMOD Browser Tools and Exercises Workshop
Part of the Next Generation DNA Sequencing Analysis Satellite Meeting
3-4:30pm, 20 March 2010
Sacramento, CA, USA
- 1 Workshop
- 1.1 From the morning session
- 1.2 GMOD Genome Browser Tools
- 1.3 GBrowse
- 1.3.1 About
- 1.3.2 Navigation and Searching
- 1.3.3 Uploading user data
- 1.3.4 Sharing tracks with other users (customers)
- 1.3.5 Securing GBrowse and its data
- 1.3.6 Advanced topics
- 1.4 JBrowse
- 2 Conference Talk
From the morning session
A Note about the GFF from the morning session:
The first column of the GFF indicates the reference sequence for the features in the GFF, and it has to match up with the reference sequences that the browser knows about. Make sure that the first column of your GFF file says NC_003070.9
GMOD Genome Browser Tools
GBrowse is the most widely implemented genome browser in use today, with several hundred servers around the world. For this tutorial, we created a server at http://gbrowse.gmod.org/gbrowse2, and specifically, a GBrowse instance of A. thaliana Chromosome 1. You might want to open that in another tab now to follow along and try things out during this section. This is another link to a similar site on the same server if the above one is giving you problems: Slower Chromosome 1.
The primary way people “get into” GBrowse is by searching for a gene or other keyword. To search, the user just enters the text (gene name or other identifier, or something in the description) into the “Landmark or Region” box at the top of the display. Since we have arabidopsis data, we could try things like “AT1G01930” or “Chr1:100000..200000”. We can also do wild card searches, like “AT1G019*”, which will give a list of matches that we can choose from.
Once looking at the GBrowse display, there are typically two or three graphical sections: the overview, region and details panel. The region panel is optional (and can be configured to be on or off by the administrator) and shows a “neighborhood” overview that doesn't cover as much area as the overview (which is always the full length of the chromosome or contig being looked at). While GBrowse has left and right pan buttons on the side of the details panel, and similar buttons along with a zoom menu above the overview panel, most people navigate GBrowse by click and drag selection on one of the scale bars for the chromosome at the top of each of panels. By pressing down on the left mouse button and moving the mouse over the region to be navigated to, GBrowse will either automatically go to the region selected, or depending on how GBrowse is configured, will pop up a menu to let the user choose what action to take. For example, in a sample yeast data set that comes with GBrowse, this is the menu that is give when the user selects a region in the details panel:
- When new tracks are selected from the ones available in the list below the details panel, the new track is added without having to reload the page
- Since the tracks are drawn separately, the can also come from different servers, giving GBrowse the ability to work with multiple rendering and database servers to make it faster than it would otherwise be with just one server.
- The tracks can also be moved up or down relative to other tracks just by clicking on the name of the track and dragging it up or down.
Uploading user data
Several file formats are supported for upload to GBrowse:
Files that are uploaded in this way are secure, in that they can only be seen by the person who uploaded them (or more specifically, on the computer where they were uploaded), and with people that they have been explicitly shared (see the section below for more on that).
In the morning section of the tutorial, we created GFF (not GFF3), so we'll use that as an example of how to upload data. (This marks the first time the server we are using is being used by a classroom—so let's hope there are no fireworks!)
To make this work, you need to have the GFF file you created in the morning session on your computer.
Go Here and find your profile, then find the GFF file from this morning and save it on your computer.
Once you done that, go to http://gbrowse.gmod.org/fgb2/gbrowse/arabidopsis. Near the top of the page, there is a link for a tab called "Upload and Share Tracks." Click on that and there is a simple interface for uploading supported files. Clicking on "From a file" will give a normal "browse" button to find the file on your computer. When you select the file and click the upload button it will, it will show a spinner while it uploads, and then give you a status report (the size of the file uploaded, and links to edit the uploaded file and the created configuration). It will also automatically load the track into GBrowse, and clicking on the Browser link will take you back to the browser display. If you see the track, but no features in it, be sure to navigate to where in the genome the features are. Try this link if you don't know where to look:
Going back to the upload tab, you can click the "edit" link for the configuration and see some of the adjustable parameters to make it look different, by changing things like the fill color (bgcolor) or the picture used for the features (glyph). For a list of commonly used parameters, take a look at:
If you run into problems and want to start over, you can use this link:
which will throw away the uploaded file and restore the default settings.
A few comments about the GFF produced this morning
The GFF produced this morning looks like this:
NC_003070.9 cashx_gff ABRF_Test 1455221 1455257 . - . ABRF_Test;Note Mismatch=0 NC_003070.9 cashx_gff ABRF_Test 1200379 1200415 . - . ABRF_Test;Note Mismatch=0 NC_003070.9 cashx_gff ABRF_Test 1170111 1170147 . - . ABRF_Test;Note Mismatch=0 NC_003070.9 cashx_gff ABRF_Test 1017175 1017211 . - . ABRF_Test;Note Mismatch=0
This is a "flavor" of GFF2 that GBrowse will accept, but doesn't really care for (yes, you may have hurt it's feelings :-)
The problem is that the first entry in the ninth column is identical ("ABRF_Test") and this tells GBrowse that these things should all be grouped together (in fact, that is called the group column in GFF versions before GFF3). As a result, GBrowse draws lines connecting all of these features as though they were all HSPs and part of one big hit in a BLAST result, and this is probably not what is intended. Two ways to improve upon this is to produce slightly different GFF2 or produce GFF3 (which has a better, more clearly defined specification).
NC_003070.9 cashx_gff ABRF_Test 1455221 1455257 . - . ABRF_Test read1;Note Mismatch=0 NC_003070.9 cashx_gff ABRF_Test 1200379 1200415 . - . ABRF_Test read2;Note Mismatch=0 NC_003070.9 cashx_gff ABRF_Test 1170111 1170147 . - . ABRF_Test read3;Note Mismatch=0 NC_003070.9 cashx_gff ABRF_Test 1017175 1017211 . - . ABRF_Test read4;Note Mismatch=0
- Added an unique identifier after the "class" (the first word in column nine).
NC_003070.9 cashx_gff match 1455221 1455257 . - . ID=read1;Note=Mismatch%3D0 NC_003070.9 cashx_gff match 1200379 1200415 . - . ID=read2;Note=Mismatch%3D0 NC_003070.9 cashx_gff match 1170111 1170147 . - . ID=read3;Note=Mismatch%3D0 NC_003070.9 cashx_gff match 1017175 1017211 . - . ID=read4;Note=Mismatch%3D0
- Third column (type) changed to match, which is a Sequence Ontology term.
- Ninth column reformated to fit GFF3 specification, including:
- URL escaping the equals sign in the Note ("%3D" is the code for equals sign).
- Used unique IDs for each of the reads.
In addition to showing the individual reads with mismatches highlighted, it will also show coverage plots as density plots (shown in this screen shot) or as xy plots, and will show read pairs, highlighting unpaired reads.
Sharing tracks with other users (customers)
Any track (one that is "part of" the server or uploaded by a user) can be shared with other users, in two different ways. One is to get a URL that can be mailed to a user to click on it and be taken to the same view that the sharer sees, and the other is to "export" the track so that it can be viewed in a GBrowse instance on another server.
To share a track, you can click on the concentric circle icon in the track name (it looks like an RSS feed icon). This will pop up a window that offers two URLs. The first one is the one we are currently interested in. If you copy that url, it can be sent in an email to someone else, can click on it and see the same track you are looking at. The url will look something like this:
This can also be done in a secure way (password protected), but the gbrowse.gmod.org server isn't set up for that. See the section below for a description of that.
This is a little more sophisticated, and we can't see an example of it in this course, but had the GFF that was created in the morning session used "Chr1" instead of the NCBI id, we could have exported our track and viewed it in TAIR's GBrowse instance. What this does is to harness the remote server (in this example, the one at TAIR) to display any of the annotations they have with our features included in the same context. This can be a powerful "mash up" used for discovery of new features.
Securing GBrowse and its data
If you have data that you would like to make permanently available on your GBrowse instance, but don't want the whole world to see it, there are several approaches that used in combination, give considerable flexibility for securing GBrowse. Some of these set ups are moderately complicated, though a senior in computer science should be able to do it with little or no guidance.
Keep it on the intranet
If you institution has an intranet, GBrowse can be kept on the intranet so that outsiders can't see it. This is a fairly simple solution if "outsiders" are the problem.
Use Apache security
The Apache web server can be configured to prompt a visitor to your site for a user name and a password, which you would only give to trusted users. This sort of security can also be configured to only allow connections from specific computers, or computers from a specific site or range of IP addresses.
Use Apache-style security on a per track basis
GBrowse also supports Apache style authentication on a per track basis, so you can specify the same sorts of restrictions mentioned above for individual tracks. Say for example you had a NGS run and the results could only be viewed by one lab. You could specify that only the computer in that lab would be allowed to see the results. You can do this for as many tracks as you like, so you could have multiple labs looking at the same GBrowse instance, but only seeing the tracks they are allowed to see.
Use GBrowse user accounts
GBrowse now also supports user accounts; you could have your users create accounts on your GBrowse instance, and let you know what the usernames are, then you can put data in their user account which would let them see their data as well. This is functionally equivalent to the Apache-style per track security, but perhaps a little easier on the GBrowse administrator by pushing some of the work off to the users.
Installing and configuring GBrowse
Installing GBrowse is relatively simple on Linux or Mac OS X servers. It is a little more challenging on Windows servers, but it can still be done. Typically, a computer science undergrad can do it in half a day, or a computer savvy biology graduate student can do it in a little longer, perhaps a day. There are a few hang ups that people run into typically relating to getting a few of the prerequisites installed.
Getting it completely configured can be quick or take a long time depending on the level of complexity desired. Typically, people take an iterative approach, getting simple things done quickly and then working on more complex things as time allows. There are a few good resources for people new to installing and configuring GBrowse:
- The GMOD wiki
- The GBrowse mailing list
- A few courses where GBrowse is covered:
- The Programming for Biology course at Cold Spring Harbor Laboratory
- Courses put on by NESCent (previously referred to as summer schools, but since this year, one is in May, and another will probably be in the fall, we have to come up with a new name). The next course, this May, is already sold out. There will likely be one next October in Japan in conjunction with the International Society of Biocuration meeting.
|More functionality||Faster, smoother UI|
|Does work on server||Moves work to web browser|
|Same underlying Perl machinery|
|Same data sources|
The moving parts in JBrowse:
You have your own JBrowse instance on the server:
SSH into the ABRF jbrowse machine: abrf2010.jbrowse.org
User: your profile name from the morning session
Copy your GFF file from this morning to this machine:
wget --http-user=abrf_workshop --http-password=abrf_HTS gff_file_url
Go into your JBrowse installation:
Add the GFF file to your JBrowse instance:
bin/flatfile-to-json.pl --gff2 gff_file.gff --tracklabel "ABRF_Test" --key "ABRF Test" --cssclass feature3
Seeing the Forest and the Trees: Visualizing Next Generation Sequence Data, by Mitch Skinner
- Part of the Session Tools to Facilitate Management, Analysis and Visualization of 2nd Generation Sequencing Data
- 3:45pm - 5:00pm, Monday, March 22