PolyPhred is a program that compares fluorescence-based sequences across traces obtained from different individuals to identify heterozygous sites for single nucleotide substitutions. PolyPhred is not a stand alone application. PolyPhreds functions are integrated with the use of three other programs: Phred, Phrap, and Consed. PolyPhred identifies potential heterozygotes using the base calls and peak information provided by Phred and the sequence alignments provided by Phrap. Potential heterozygotes identified by PolyPhred are marked for rapid inspection using the Consed tool.
To run this software interactively in a Linux environment run the commands:
module load polyphred polygen
Running the PolyPhred: Phred, Phrap, Consed and PolyPhred all require a fixed directory structure for analyzing sequence data. If a gene to be analyzed is called "mygene", for example, the directory structure should look like this:
containing the subdirectories:
/cchromat_dir /edit_dir /phd_dir /poly_dir
1. For each set of data to be analyzed, create this directory structure.
2. Move or copy the chromat (trace) files to the chromat_dir directory.
3. From the edit_dir directory, run "phredPhrap mygene" where "mygene" is the name of the data to be analyzed. This program runs the programs Phred and Phrap consecutively. When the process is complete, there should be several files in the edit_dir, including one with the extension .ace.1 (the ACE file), and several files in the phd_dir and poly_dir directories.
4. View the assembled sequences in Consed. Further assembly of the data might be required. For information on this process, check the Consed documentation.
5. Run "polyphred". Include any desired flags on the command line.
6. Use Consed to view the polymorphic sites.
For additional information
- Documentation can be found at http://droog.gs.washington.edu/polyphred/poly_doclist.html
- Home Page: http://droog.gs.washington.edu/polyphred/