PLPTH 613
Bioinformatics Applications
Spring 2009


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Lab 5. Assembly of fingerprinted clones

Introduction

In the last lab we practiced doing assembly of sequenced subclones based on alignment. In this lab we will practice assembly of the superclones (in plant genomics, these are usually BACs) of which these subclones are fragments. So Lab 3 and Lab 4 are in reverse order to reality, and I hope this doesn't confuse you. In a real genomics project, today's operation would precede subcloning and sequencing. We would not select BACs to sequence until we had optimized BAC selection by producing a minimum tiling path from a scaffold of fingerprinted BACs. This is what we will do today, with the FPC (FingerPrinted Contigs) program:
  1. Load a set of files describing restriction-fragment sizes from digestion of a set of BACs (= clones)
  2. Identify contigs based on the Sulston coincidence score calculated for each pair of clones
  3. Attempt to resolve spurious connections
  4. Add marker data to anchor clones to chromosomes
  5. Identify a minimum tiling path (MTP)
  6. Become more familiar with making XWindows connections to a remote computer
  7. Become familiar with FPC's screen displays.

Setting up an XWindows connection

XWindows is a protocol that allows a description of a graphical display on one computer to be sent to a remote computer and drawn on that computer's screen. We typically use XWindows to run Unix or Linux applications while controlling them from a graphical user interface (GUI) that appears on our remote PC's screen. We do this through an XWindows server (ours is called Xming) and a slightly modified putty connection. Here are the instructions for making the connection and starting FPC:
  1. Locate Xming on your computer and start it.
  2. Locate putty and start it. Set up a New session with an IP address that you'll be given, set X11 forwarding as instructed in last lab, and click Login.
  3. Enter user name bioinfappl613 (Enter) and the password that you'll be given (Enter). Once you see the command prompt indicating a successful login, type fpc and press Enter. You should see a new window called FPC V9.3 Main Menu.
  4. Note that for security, this password will be changed in a week. You may be able to finish the lab today; if not, you may work in the lab again in the days until the next lab. Don't try to run an XWindows session over a slow connection, since the necessity of sending graphics-updating instructions over the Internet causes a severe lag in response times.

Running FPC

  1. You may work alone or in groups of two. You may discuss the questions to be answered, but each student must still turn in a separate report.
  2. We will follow tutorials provided by the FPC developers. For definitions of terms you don't understand, refer to this help document. It's convenient to have the tutorial page open on one screen and the FPC application running in a different one.
  3. The terminal window that you opened in order to start FPC can't be used for typing commands until you quit FPC. In the meantime it will be used by FPC for displaying progress messages. To interact with the host computer, you may open a second putty connection, but in this one don't start fpc.
  4. Some starting questions for which you'll need to find out the answers:
    What are Q clones?
    What is a     CB map and how does it differ from a physical map created by sequence alignment?
    What are     buried clones?
    What are     chimeric contigs?
  5. Start with the main FPC tutorial. We covered in lecture the main ideas of the analysis, so go straight to the section entitled Building a physical map with FPC.
  6. Work your way through this tutorial. Note that you can ignore the section Searching and the material about adding remarks, although you will need to add markers in that same section. Also note that sometimes when the tutorial says to "select" a label, you actually need to double-click on it in order to make a window appear. Finally, don't close windows as you would in Windows, using the X box. Instead, right-click in a white area of the window and choose Close from the pulldown menu.
  7. In the Merging Contigs section, the documentation is a bit thin. What they mean is that for two contigs to be merged, the right end of the first one must be next to the left end of the second one; that is, they must be in RL orientation. If the first contig is labeled L in the results of Ends-Ends, it will need to be flipped; likewise if the second is labeled R, it will need to be flipped, and you, the analyst, must do this. Makes you wonder why the program can't work this out for itself.
  8. In the same section appears the following statement. In your report, explain this statement: The reason the CB map is not automatically recomputed is that manual merges generally are found at less-stringent cutoffs, and assembling even a good contig at a less-stringent cutoff can result in errors, since clones may have false-positive overlaps with other clones in the same contig.
  9. In merging contigs, the tutorial instructed us to change the setting of Match from 2 to 1 in the Main Analysis window. What would you expect to be the effect on the End-End merge test of leaving the setting at 2?
  10. Viewing the Ctg1 window, select marker F100. Why do you think only two clones are highlighted (showing that they hybridize with this marker) even though these clones show overlapping fingerprints with many other clones?
  11. View the fingerprints of these two clones (C1114F11* and H0210F04*) by right-clicking one and selecting Fingerprint from the pulldown menu. Select the Add button and then add the second to the Fingerprints viewer. Use the Zoom: In button, and the slider on the left, to magnify a pair of bands (one red, one blue) at around 672, or 768. Change the number next to the Tolerance... button to a smaller value and press Enter. Do this until the colors of your bands turn to black. Explain why this caused FPC to change the colors. You may then close this window.