Phred is an algorithm developed by Phil Green's group at the University of Washington. Phred re-evaluates the chromatogram peaks in a trace file, a process known as "Base Calling". Not only is phred typically more accurate than the default base callers used by automated sequencing machines, but it also assigns "Quality Values" to each individual base call. Phred uses a statistically significant logarithmic scale from 0 to 99 where 10 means there is a 1 in 10 chance that the call is in error, 20 means there is a 1 in 100 chance the call is in error, 30 means there is a 1 in 1,000 chance of an error etc. The values 98 and 99 are reserved to indicate residues that have been edited by the user. A phred score of 20 or more is generally considered to be an acceptable score. MacVector Assembler displays phred scores as a histogram above the sequence using colors to indicate the quality - scores below 20 are shown in red, scores of 20 or greater in green and edited residues (score 99) in blue.
Make sure you have no selections in the project window. To toggle a selection off, click on the selection while holding down the command ("Apple") key.
You can select a subset of sequences for analysis if you wish. However, if nothing is selected, all of the chromatogram sequences in the project will be submitted for analysis.
Choose Analyze | Base Call (phred).
A progress dialog will appear. You can close the dialog if you wish and follow progress using the Job Manager.
When the job has completed, the project window will refresh to reflect the new base calls. The status of each entry changes to "P" to indicate you have run phred on the sequence.
Double-click on one of the phred-called sequences in the project window to open up the trace editor window.
When you open the trace editor window from an assembly project, two additional toolbar buttons are displayed.
Show Qualities - toggle this button to show or hide the quality histogram displayed over the top of the sequence.
Show Base Calls - toggle this button to show or hide the basecalls displayed immediately below the main sequence line.
You can see that the original base call for this sequence was generated using a utility called "makeSCF". The phred base call is shown directly underneath this. You cannot edit the base calls - they are read-only. You can edit the upper sequence if you wish - this is the "active" editable sequence that is used in all assemblies and analyses. If you subsequently re-run phred on a sequence, it will replace the phred base call and will also replace any edits you have made to the active sequence.
The phred.pdf file in the MacVector/Documentation/ folder contains the original University of Washington documentation for the phred algorithm.