As a general rule, you should use "Call Bases" only if you have sequences that do not have quality scores. If your sequences already have quality scores, the "Call Bases" function should be used only in special circumstances.
Here's why:
The "Call Bases" function in CodonCode Aligner uses the base calling program PHRED to replace the existing bases, and assign quality scores to all bases. This is useful because CodonCode Aligner will work much better if sequences have quality scores. However, most sequences that you get from ABI sequencers nowadays already have quality scores. These are produced by ABI's base calling program, the "KB basecaller". In general, the KB basecaller will be more accurate than PHRED, and give more accurate quality scores. If you use the "Call bases" function in Aligner for sequences that already have quality scores from the KB basecaller, this will probably result in more errors, and less accurate quality scores. While the differences are small for many sequences, in some cases, PHRED results will be a lot worse; for example, PHRED may make obvious errors in perfectly good sequence, or assign quality scores that are clearly much too low.
Why does PHRED make these errors? Simply because PHRED was developed a different kind of data - sequences produced on ABI 373 sequencers, not the modern capillary sequencers. When capillary sequencers like the ABI 3730 were introduced, development on PHRED had pretty much stopped. But at that time, ABI finally got their act together, and produced a base calling program that also assigned accurate quality scores (the KB basecaller). In the past few years, ABI has continued to adapt the KB basecaller to new sequencing machines and chemistries, while the PHRED developers had gone on to do other things (like being the first to discover that the human genome contained much fewer genes than everyone had anticipated).
How to tell if your sequences already have quality scores:
After adding ABI files to a project in CodonCode Aligner, you can simply check the "Quality" column in the project view. Sequences without qualities will have a "0" in this column. If you see other numbers, the sequences already have qualities.
Exceptions: when to use "Call Bases"
- If your sequence do not have quality scores, for example because they are really old, or the sequencing facility does not use the KB basecaller.
- If you have sequences from non-ABI sequencing machines (MegaBACE, Beckman, ...) where PHRED may give better results than the basecalling program that was used originally.
- If you really know what you are doing, and you know for a fact that PHRED works well on your data.