Alignments for Clonotypes

The alignments view (Image assembly_editor_16_n_p) shows all reads mapping to a specific clonotype (figure 7.14).

Image clonotype_alignment
Figure 7.14: Read mapping for a TRB clonotype. V, D, J and C segments are annotated on the reference sequence and the CDR3 is annotated on the consensus.

The alignment contains:

Various settings can be configured in the side panel, see

For further processing, the alignments can be opened and saved as a stand-alone read mapping by using the "Open as Read Mapping" button.

The clonotypes for which the alignment should be shown can be selected from the drop-down menus in the side panel, or from the clonotype table (Clonotypes Table) while using a split view, see figure 7.15.

Image clonotype_alignment_split_view
Figure 7.15: Clonotypes split view. Top: Multiple clonotypes sharing the reference segments are selected in the table view. Bottom: Alignment view for the clonotypes selected in the table view.

Alignments for multiple clonotypes can be shown together provided that they have the same V and J segments and the D / C segments are not contradictory: either the D / C segment is identified and the same, or it is missing (figure 7.15).

When viewing alignments for multiple clonotypes, it can be useful to change "Compactness" to "Not compact" and tick the "Clonotype #" annotation from the side panel. This way, it is easy to see the clonotype assigned to each read, figure 7.16.

Image clonotype_alignment_annotation
Figure 7.16: Alignment view for multiple clonotypes where "Compactness" is set to "Not compact" and "Clonotype #" is ticked.

Figure 7.15 shows an alignment for two clonotypes. Some of the reads do not span past the J segment and using the "Clonotype #" annotation, we can confirm that these reads belong to clonotype # 400, which is the clonotype without the C segment (figure 7.16). The two clonotypes do not share the D segment and have different CDR3 sequences. Using the alignment view (figure 7.15), it is straightforward to spot the differences between the two CDR3 sequences.

Alignments after merging

Alignments for clonotypes with ambiguous segments are calculated using the first segment in the list. Thus when ambiguous segments have been resolved using Merge Immune Repertoire, some reads may not be included in the alignments view. This happens when the unique segment that the ambiguous segments resolved to was not the first segment in the list.

Two situations can arise as illustrated in figure 7.17:

Image incomplete_alignments
Figure 7.17: Clonotypes split view. Top left: Table view of TRG clonotypes that have been merged. Top right: Alignment view for clonotype 3. The alignment could not be determined and the view shows an alignment of coverage 1. Bottom: Alignment view for clonotype 7. The clonotype count is 135, while the alignment coverage is only 126, i.e. the alignment for 9 reads could not be determined.