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 (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