Maloof Lab:eBioX

eBioX is a free Mac DNA sequence viewer and editor that provides a handy interface to alignment and assembly programs.

Prerequisites

• eBioX can be downloaded from http://www.ebioinformatics.org/ebiox/
• You will also need eBioTools which includes pre-compiled versions of many helpful alignment and analysis programs
• The computer should have X11 installed (standard on Leopard)
• eBioX has been installed on the lab computers "June" and "Tallac"

Alignments

• You can open a .txt file containing multiple fasta (wikipedia:FASTA_format) sequences. Other formats may work also; I have not tried.
• Or choose New Alignment from the File menu and drag or choose the files.
• From the Alignment menu choose your favorite alignment algorithm. Alignment>Align All>Muscle is a good place to start.

Viewing individual trace files

• eBioX can read .ab1 or .ztr files.
• Chose open from the file menu (or type CMD-O) to open the file.
• Use the zoom slider to adjust the view
• Drag with the mouse to select the good range of sequence.
• You can then extract the good sequence by pressing the "extract" button. This extracted data will need to be saved into a new file.

Assembling multiple sequence trace files

• Use eBioX to open programs in the STADEN package. The STADEN programs run in an X11 window.
• Use Pregap4 to automatically clip your trace files to retain those bases with a minimum quality score that you determine, remove vector sequences, etc.
  • From the tools menu in eBioX choose Staden>Pregap4
  • click on the add files button and add your files. Holding down the ctrl key allow you to select multiple files.
  • click on the Configure Modules tab and choose your options. Clicking on an individual option (ie Sequencing Vector Clip) allows you to set parameters for that option.
  • click on Run; check the textual output.
  • If you have no more files to process chose exit from the File menu.
• Use gap4 to assemble your clipped sequence files into a contig. WARNING: You will get errors if there are any spaces in the path (any directory/folder) leading to your sequence files, or in the filenames themselves.
  • From the tools menu in eBioX choose Staden>gap4
  • You first need to create a database file that will keep track of which files are in the contig. Choose file>new. Click on the browse button to navigate to the directory where your sequences are (or for a large project maybe one directory above). Enter a filename for the database.
  • Next choose Assembly>Normal shotgun assembly.
    • select Input reading filenames from selection
    • click the browse button and select your files. (Chose the .exp files that were output from Pregap4)
    • select a list or file to save the names of failures to. (you can access lists later from the Lists menu).
  • You may want to include a backbone sequence to assemble onto (if available). This should be a .txt file
  • You can view your new contig by selecting view>Template Display from the main gap4 window. I find it best to deselect Templates in the subsequent dialog box. Double-clicking in the resulting window will bring up a new window showing the actual sequence and consensus. You can edit the alignments and consensus from this window. You can save a consensus file either from this window or from the file menu.
  • There are many other useful functions and opens, so go exploring. The view>Find internal joins can be particularly useful for difficult assemblies.
• For more help on Staden see http://staden.sourceforge.net/documentation.html