DNPTrapper Manual and Tutorial

Quick Start

Overview

Concepts

Directories

Supported file formats

Views and windows

Moving modes

Zooming

Context menu

• Operations

• Compact Reads - reorganizes the reads to occupy as little vertical space as possible.
  
• Export Consensus - writes the consensus (according to majority vote) to file.
• Find DNPs - analyzes selected reads for DNP content.
• Find Mates - finds and selects mate pairs of selected reads, if such data has been imported.
• Quality Trim seqs - marks up low quality ends on reads. Low quality parts will be shaded, and will be excluded from some operations (e.g. Find DNPs).
• ReAlign - locally optimizes alignment of selected reads.
• Remove DNPs - removes DNPs in selected reads.
  
• Remove Quality trimming - resets low quality mark up.
  
• Search reads for DNP ID - highlights reads containing a certain DNP.
• Select reads from file - highlights reads specified in a file with readnames.
• Select strand - highlights reads of specified strand (normal or reverse).
• Sort according to DNPs - a DNP grouping algorithm. Naive, but pretty useful as a starting point.
• Write ACE file - writes selected reads to a file in the ACE format.
• Write alignment to file - writes alignment in ASCII format to file "alignment.out" in current directory.
• Write readnames to file - writes the names of selected sequences to a file. Very useful for saving different selections and used in combination with "Select reads from file" above.
  
• Write selected seqs to file - writes the selected sequences, and their corresponding quality values, to file in FASTA format.
  

• Info

• Read info - read name, strand, quality region, local position in read and list of read's DNPs.
• Feature info - base, quality value and DNP ID, if any.
• General info - row and column  in alignment.

• Switch to viewmode

• Chromatogram options

• Set Chromat center - aligns chromatograms at cursor position and highlights the column with a vertical line. NB: This can be used also when no chromatogram data is present and can be useful for keeping track of the column currently being investigated.
• Clear Chromat center - clears chromatogram alignment position (i.e. sets it to 0).

• Scroll here

Menus

Contig

        Open

        Close

        Exit

Edit

        Undo last move

        Cut

        Copy

        Paste

        Select All

        Select Between Rows

View

        Enlarge

        Shrink

        Show statistics

Window

        New view

        Cascade

        Tile

Tools

        Import project

        Import Chromatograms

        Import Mates

        Import phd data

        Normal Mode

        Drag Mode

Chromatograms and phd files

Mate Pair data

Native file format

Safety, backups etc

Known bugs and problems

• A minor visual bug at the lowest zoom level when scrolling.
• It is possible for the user to change directory names in the Open File dialog. Dangerous!
• ReAligning takes quite a while to perform currently, a faster implementation is under way.
• Dates etc in exported ACE file are bogus.

Coming attractions

• Autoscrolling in normal moving mode and with rubberband
• Choosing filename in "Write to file" algo
• Exporting to ACE file format
• Better support for mate pairs
  

Tutorial

• test.seq - FASTA file with simulated shotgun reads sampling repeated sequence with 2% mean difference between any two repeat units.
• test.qual - FASTA file with corresponding quality values.
• test.ace - ACE file produced by phrap for this dataset.

Tutorial Start

1. Go to the trapper installation directory. Run the aceparser program (bin/aceparser) without any parameters to see a short help text on how it is used. Run it again with test.ace and test.qual to generate test.ace.xml. This might take a short while.
   
   
2. Make an empty directory called trappertest that will be the new project directory.
   
   
3. Open DNPTrapper: bash$ bin/trapper -d /path/to/trappertest -c config/trapperconf.xml
   
   
4. From the Tools menu, choose "Import project" and choose the generated trappertest.xml in the file dialog.
   
   
5. From the Contig menu, choose "Open" (or click the directory icon on the toolbar). A directory called "Contig0" should now have appeared in your project directory. Choose it and click OK.
   
   
6. You should now see an alignment consisting of black boxes on white background. They represent the reads in the contig. If you zoom in, you will see the base sequences and quality values visualized. Zooming can be performed using toolbar buttons or by choosing from the View menu, alternativly you can press the "+" and "-" keys on your keyboard. If you press Ctrl at the same time, the zooming is only performed in the X-direction. Similarly, pressing Ctrl + Alt zooms in Y-direction only. You can scroll using the mouse, or by using the arrow keys. Hitting Ctrl while using the arrow keys speeds the scrolling up.
   
   
7. Try selecting and un-selecting some reads by Ctrl-clicking them. Also try the "rubber band" functionality for read selection by pressing the left mouse button where it doesn't hit a read and drag the mouse pointer. Select all the reads by either a) choosing "Select All" from the Edit menu, b) hitting Ctrl + A on the keyboard, or c) pressing the "Select All" button in the toolbar. If you click where no read is located, all reads are de-selected. Right click on a read and get familiar with the different options in the context menu that pops up (see above for more detailed description of this menu).
   
   
8. As you notice when you zoom out, each read is located on a separate row. This makes it hard to get an overview of the contig, so we'd like to reorganize the reads to occupy less vertical space. Select all reads, then right click on one of the selected reads and choose "Compact Reads" from the Operations entry in the context menu. You will be asked to decide at what row the compacted alignment should start - choose row no. 1.
   
   
9. Look around in the alignment by zooming in and out, scrolling etc. If you look closely, you'll see that the alignment isn't locally optimal everywhere. This is because phrap doesn't optimze the alignment. To optimize the alignment, choose "ReAlign" from the Operations option in the context menu. This will take a few seconds.
   
   
10. Now it's time to try separating repeats. Make sure all reads are selected and run the "Find DNPs" operation from the context menu. This takes a few seconds. For more information about DNPs, see these references.
    
    
11. As you see, a lot of dots with different colors appear in the alignment. They represent DNPs. Let's try some different ways of resolving the repeats.
    
    
12. Select one read containing DNPs and drag it below the alignment. Using your eyes, try to spot other reads containing DNPs with the same color on the same column. Drag them down too. If you see "new" DNPs to the left or right of the first one, look for them in the alignment also and drag the reads down to fit with your current working group. As you notice, you're only allowed to drag the reads vertically. This makes sense, since you want to keep the bases aligned properly. Try this for a while in order to get a feel of how you can organize the reads into different repeat groups.
    
    
13. As you notice, this is a slow and tedious way of doing things. Let's try a faster version of the same method. Compact the reads again (see step 9). Again, choose a read containing DNPs and drag it below the alignment. Choose a DNP and find out it's ID by right clicking it and looking it up under "Feature info" under the Info subsection of the context menu. If you don't see it, it's because you didn't hit it, which could be due to the fact that one pixel corresponds to several positions when you're zoomed out. But not to worry. If you look under "Read info", you'll see that all DNPs are listed for the read along with their positions and IDs. Also listed is the current position in the read. The DNP you're interested in is probably located quite near this position. Remember its ID.
    
    
14. Now select all reads above the DNP you chose to work with, by dragging the "rubber band" so that it intersects all reads at that position, or by choosing "Select between rows" from the Edit menu and typing in 1 and a large number as parameters. Right click on one of the selected reads and choose "Search reads for DNP ID". In the query box that pops up, type in the ID for the DNP you're working with. As you see, the reads that have this DNP will remain selected, while the others are un-selected. Now you can simply press one of the selected reads and drag all of them to the read you started with.
    
    
15. Choose an other DNP in your current "working set" by repeating step 14 and 15, but instead of dragging the reads manually to your group, use the "Compact Reads" operation in order to put them in a nice compact fashion starting on a row just below your group.
    
    
16. Now let's try the fastest method of resolving the repeats. Again, compact all the reads (see step 9). Make sure all reads are selected and run "Sort reads according to DNPs". You'll be asked for a starting row, choose no. 1. As you see, the repeats organize themselves into fairly neat piles with similar DNP content. In some ambigous cases the algo will make a wrong choice, but that's quite easy to correct manually using the techniques from the previous steps.
    
    
17. Finally, it's time to try to connect the different repeat groups. It is easy to verify that phrap has resolved the repeats partially. It can be done e.g. by exporting the consensus sequence ("Export Consensus" under Operations) and viewing a dot plot of it against itself using a suitable tool (e.g. Dotter). Actually, phrap has separated the repeats into two units. This means that some DNPs should be present in two versions, one on the left hand side of the alignment and one on the right hand side. This is the case for some DNPs in this example. The DNP colors reflect what type of DNP we have. For instance a red DNP means that the DNP is an A, while the consensus on that column is T. See below for complete color code of DNPs. In our example, some DNP patterns can be detected visually. This can be a bit hard to see, so you get a hint: find and look at DNP IDs 18 and 23. They correspond to DNP IDs 549 and 550 both in color and in spacing. Verify this.
    
    
18. Create a new contig using "Open". In the file dialog, create a new directory and name it (e.g. to NewContig). Choose it. Under the Window menu, choose "Tile". Now you see the new contig and the old one next to each other. If you think it's more convenient, you can manually arrange the contigs on top of each other instead. Now select all reads in the group containing DNPs 18 and 23. Choose "Drag Mode" from the Tools menu. Now you can drag the selected reads into the new contig. This copies them to the new contig, so they're still left in the old one - if you want to remove them, you have to use "Cut".
    
    
19. Repeat the above step for the group containing DNPs 549 and 550. Try to align the matching DNPs against each other. If you zoom in, you'll see that they don't match exactly(there are some gaps in one of the groups that displace one of the DNPs), but if you ReAlign you'll see that they match perfectly. Presto!
    
    

References