Sequencher 4.8 User Manual--PC - Bioinformatics and Biological ...

absolute number of bases. For example, you could specify that fragments must have
fewer than 3 N’s in the first 20 and last 30 bases. To activate specific trim criteria you
must first select the checkbox that governs the criteria fields.
Figure 6-3 The Ends Trimming Criteria window
Using Confidence to Trim Ends
In addition to trimming data based on the number of ambiguities at the 5’ and 3’ ends,
you can trim sequence data based on the confidence scores assigned by your base caller.
Confidence scores (also called quality values) are numbers associated with each base call
and which define the likelihood that a base call is incorrect. The most common scale is
from 1-60, where “60” represents a 1/10 6 chance of a wrong call and 20 represents a 1/10 2
chance. Depending on the program used, the confidence score may be based on peak
height, the presence of more than one peak, and/or the spacing between the peaks. (See
Figure 6-3 the Ends Trimming Criteria window for trimming by confidence criteria.)
Reviewing the Trim
Each pane in the trimming windows shows schematically how much sequence should
be trimmed off. Blue lines represent the acceptable sequence data. The blue line is then
flanked by red scissors, which mark both the 5’ and 3’ suggested trims based on the
current Trim criteria. The remaining red regions of the sequence define the portion of the
sequence to be trimmed.
Click the Show Bases button to see more information on the bases. After you have
clicked Show Bases, the button toggles to Show Overview. Sequencher also provides
SEQUENCHER 4.8 User Manual for Windows © 1991 - 2007 Gene Codes Corporation, Inc. All rights reserved.
35