Fine-Tuning Mutation Detection with CodonCode Aligner

CodonCode Aligner includes several preferences and display options that let you control how mutations are detected, displayed, and reviewed. This page explains the most important settings and where to find them.

Mutation Detection Settings

To access the settings, choose Preferences from the Edit menu (on Windows) or Settings from the CodonCode Aligner menu (on macOS), then select Mutations in the sidebar.

The mutation detection preferences dialog looks like this:

Detection Sensitivity

Use this section to control how aggressively Aligner identifies heterozygous point mutations. There are two separate sensitivity settings:

• With intensity drop — used when a secondary peak is accompanied by reduced primary peak intensity.
• Without intensity drop — used when there is no clear drop of intensities, for example because all samples at this position are heterozygous.

Higher sensitivity detects more variants but may increase false positives due to noise. Lower sensitivity is more conservative but is more likely to miss some heterozygous point mutations, for example if the secondary peak is weak. The medium setting is recommended as a starting point.

Data Quality Filtering

Low-quality ends of traces often contain noise. You can control how much of each sequence is excluded from analysis by adjusting the Minimum quality at ends value. Higher values (e.g., 40) exclude more bases, reducing false positives at the expense of possibly missing mutations in lower quality regions at the start and end.

Excluded regions are marked with dataNeeded tags (typically shown as yellow boxes, depending on your highlighting settings).

When the Use noise filter tags checkbox is selected, additional analysis of primary, secondary, and tertiary peaks in all samples at a given position is used to reduce the number of false positives from "noise" peaks in traces, possibly at the expense of increasing false-negative errors.

Marking Mutations

This section controls how Aligner annotates mutations, and optionally edits base calls:

• Add tags only to mutated bases — limits tags to sample bases that differ from the consensus, or have a heterozygous mutation. Useful when focusing on rare variants.
• Use ambiguity codes for heterozygous point mutations — replaces heterozygous base calls with IUPAC codes (e.g., "R" for A+G).
• Change homozygous mutations to lower case — converts calls at homozygous mutations to lower case for easier downstream analysis.
• Remove existing mutation tags — choose whether to remove all or only unedited tags before running "Find Mutations" again.

Note: Tag edits and base call changes are not linked — if you manually change a tag, you’ll need to update the base call separately if needed.

Homozygous-Only Mode

If you're working with clonal samples where heterozygous mutations are not expected, you can enable Look only for homozygous mutations. This skips all checks for secondary peaks and uses only consensus mismatches.

Include Heterozygous Indels

By default, Aligner will also look for heterozygous insertions and deletions. You can disable this option if indels have already been detected, or to speed up detection slightly.
Tip: For best results, detect heterozygous indels before trimming sequence ends.

Feature Display Settings

CodonCode Aligner displays detected mutations and other annotations (such as coding regions or excluded areas) as visual tags. You can control which tags are shown in the views by adjusting the Features preferences.

In the middle part of the dialog, you can select other "Features or interest" that CodonCode Aligner should use for feature navigation, for example gaps of low-quality regions. When analyzing mutations, it usually is best to uncheck all these options.

The top section of the dialog lets you select which tags should be shown. To show all tags relevant in mutation detection, you can either:

• Select the "All tags" radio button, or
• Select specific tag types to be shown by pressing the "Specify.." button.

When you press the "Specify.." button, the following dialog will be shown:

You can select which tag types to show by selecting individual types in the list on the left side. To show all relevant tags for mutation detection projects, it is easiest to first press the "Select None" button at the bottom, and then select the "All Mutation tags" checkbox.

Highlighting Options

CodonCode Aligner can highlight tagged regions in the contig and trace views using boxes, background shading, or both. You can control the appearance of these highlights in the Highlighting preferences panel:

For mutation analysis projects, it is best to use "box" highlighting for tags, since the color of the box indicates the tag type.
Note that if "None" is selected for tags, mutation tags will not be highlighted under the bases, regardless of what is selected in the "Features" settings!

Alignment Preferences

A common approach in mutation detection is to use genomic reference sequences that cover the entire gene, while searching for mutations exon by exon. In such projects, CodonCode Aligner can automatically clip the reference sequence in the aligned contigs to the aligned region. This can be chosen in the Alignment settings:

When "Clip to alignment or exon" is chosen, the reference sequence copy in the aligned contig will be clipped to the exon boundaries, or the start and end of aligned sequences, whichever extends further. You can also select to leave additional bases of the reference sequence at the start or end of the contig for additional context sequence.

When "Clip to alignment or exon" is chosen, the reference sequence copy in the aligned contig will be clipped to the exon boundaries, or the start and end of aligned sequences, whichever extends further. You can also select to leave additional bases of the reference sequence at the start or end of the contig for additional context sequence.

Consensus Method

When looking for mutations relative to a known reference sequence, make sure that the consensus method settings are set to use the reference sequence as the consensus sequence, as shown below:

Toolbar Customization

CodonCode Aligner's toolbars can be customized, which can simplify manual verification of putative mutations. For example, you can add the buttons to confirm or reject SNPs, which are highlighted in the screenshot below:

Many actions can also be started through keyboard shortcuts, menu selections, or context-sensitive popup menus to suit different user preferences.