Amino Acid Changes

This tool annotates variants with amino acid changes and creates a track for visual inspection of the amino acid changes. It takes a variant track as input and also requires a track with coding regions and a reference sequence.

To add information about amino acid changes to a variant track:

        Toolbox | Resequencing Analysis (Image resequencing) | Functional Consequences (Image functional_cons_folder_closed_16_h_p) | Amino Acid Changes (Image gg_amino_acid)

If you are connected to a server, the first wizard step will ask you where you would like to run the analysis. Next, you must provide the variant track to be annotated with amino acid changes (see figure 32.18).

Image aminoacidchanges_step1
Figure 32.18: The Amino Acid Changes annotation tool takes variant tracks as input.

Click Next to go to the next wizard step (figure 32.19).

Image refiner-aminoacid
Figure 32.19: Select CDS, mRNA, and sequence track and choose whether or not you would like to filter away synonymous variants.

Click Next, choose whether you would like to Open or Save the results and click on the button labeled Finish.

Two types of outputs are generated:

  1. A variant track that has been annotated with the amino acid changes. The added information can be accessed via the tooltips in the variant track or in the extra columns that have been added to the variant table. The extra columns provide information about the amino acid changes (see The variant track opens in track view and the table view can be accessed by clicking on the table icon found in the lower left corner of the View Area.
  2. An amino acid track that displays a graphical presentation of the amino acid changes. The track is based on the CDS track and in addition to the amino acid sequence of the coding sequence, all amino acids that have been affected by variants are shown as individual amino acids below the amino acid track. Changes causing a frameshift are symbolized with two arrow heads, and variants causing premature stop are marked with an asterisk. An example is shown in figure 32.20. The information on the individual amino acids is displayed when the box is wide enough (three bases). This is typically the case -- if not, the side panel settings can be adjusted accordingly (e.g by decreasing the "hide insertions below (%)" value). The information is always displayed in the tooltip on the box.

Image amino_acid_changes_frameshiftstop
Figure 32.20: The variant track and the amino acid track is here presented together with the reference sequence and the mRNA and CDS tracks. An insertion (purple arrow) has caused a frameshift (red arrow) that has changed an alanine to a stop codon (blue arrow).

For each variant in the input track, the following information is added:

Note that variants located at the border of exons are considered intronic (i.e. located between the last intronic and first exonic base or between the last exonic and first intronic base). Amino acid changes will therefore not be determined for these variants.

An example of the output is given in figure 32.21.

Image amino_acid_changes_linked_track_table
Figure 32.21: The resulting amino acid changes in track and table views. When the variant table has been opened by double-clicking on the text found in the left side of the View Area, the variant table and the variant track are linked. When clicking on an entry in the table, this position will be brought into focus in the variant track.

The top track view displays a track list with the reference sequence, mRNA, CDS, variant, and amino acid tracks. The lower table view is the variant table that has been opened from the track list by double-clicking on the variant track name found in the left-hand side of the View Area. When opening the variant table in split view from the track list, the table and the variant track are linked.

An example illustrating a situation where different variants affect the same codon is shown in figure 32.22.

Image aminoacidchanges_threedeletions
Figure 32.22: Amino acids encoded from codons that potentially could have been affected by more than one variant are marked with a hash symbol (#) as the graphically presented amino acid changes always only include a single variant (a SNV, MNV, insertion, or deletion). Shown here are three different variants, present only one at the time, and the consequences of the three individual deletions. In cases where the deletion is found in a codon that is affected with an amino acid change, the arrow also include the deletion (situation 1) in the two other scenarios, the codon containing the deletion is changed to a codon that encodes the same amino acid, and the effect is therefore not seen until in the subsequent amino acid.

In this example three single nucleotide deletions are shown along with the resulting amino acid changes based on scenarios where only one deletion is present at the time. The first affected amino acid is shown for each of the three deletions. As the first deletion affect the encoded amino acid, this amino acid change is shown with a four nucleotide long arrow (that includes the deletion). The other two deletions do not affect the encoded amino acid as the frameshift was "synonymous" at the position encoded by the codon where the deletion was introduced. The effect is first seen at the next amino acid position (763 and 764, respectively), which does not contain a deletion, and therefore is illustrated with a three nucleotide long arrow.

The hash symbol (#) on the changed amino acids symbolize that more than one variant can be present in the region encoding this specific amino acid. The simultaneous presence of multiple variants within the same codon is not predicted by the amino acid changes tool. Manual inspection of the reads is required to be able to detect multiple variants within one codon.

Known limitations

When two genes overlap and an insertion in the form of a duplication occurs, this duplication will be labeled as an insertion.

The Amino Acid Changes tool will not perform flanking checks for exons/CDS that wrap around the chromosome in a circular chromosome.

The amino acid track

The colors of the amino acids in the amino acid track can be changed in the Side Panel under Track layout and "Amino acids track" (see figure 32.23).

Image amino_acid_track_colors
Figure 32.23: The colors of the amino acids can be changed in the Side Panel under "Amino acids track".

Four different color schemes are available under "Amino acid colors":