Once sequences have been analyzed, alignments can be displayed to the alignment window. For each sequence, CDRs and framework regions are displayed using user-defined templates and rules, and the sequence is annotated.
The interface provides many key facilities including:
- Highlight mutations, frameshifts,
- Highlight sites of potential post-translational modification and STOP codons
- Cluster sequences by region: CDR1, CDR3…
Scaligner assigns a position to aminoacids and defines FRs and CDRs according to 6 numbering schemes: Kabat, Chothia (pre-89), Martin, IMGT, IMGT with Kabat CDR3, and AHo.
The numbering scheme can be modified after analyzing the sequence in the user profile.
We provide in this section technical information on this procedure.
CDR and FR definitions
The definition of regions is linked to the numbering scheme.
Any other arbitrary definition can be defined in the user profile and will be used globally for the authenticated user.
Germline mutations and frameshifts
As frameshifts are an important piece of information, they are always displayed in front of the sequence name with a “F” on a red background to remind that the sequence has been altered during analysis to optimize the alignment to the germline.
In the alignment window, check “Compare to germline” to display mutations and frameshifts. Mutations are displayed on a blue background, and frameshifts are displayed on a red background on each site.
Potential post-translational modification
In the alignment window, check “Highlight PTM in VH” or “Highlight PTM in VL” to display which aminoacids might be subject to PTM in either region.
The list of PTM recognized by Scaligner can be configured and it is provided in the sequence window.
As the presence of a stop-codon in a sequence is an important piece of information, it is always displayed in front of the sequence name with a “P” on a green background.
In the alignment window, check “Highlight STOP codons” to display the position of stop-codons.
In the alignment window, click on one or several regions (CDR1 in the example below). The sequences are sorted according to the selected region(s).
Sequences that are identical on the selected region(s) are grouped in clusters. Clusters are identified by coloring sequence names with the same background. In the example below, sequences BSA3, DPL16, Humlv418, PR-TS2, THY-29, mAb63 and E29.1 LAMBDA share the same CDR1.
The number of clusters is given “57 unique” in this example.
Scaligner offers also clustering capabilities based on similarity threshold, and not only on sequence identity.
In the alignment window, select a sequence (Humlv318 in the example below), then click CDR2 and CDR1 while pressing the Ctrl key.
Sequences are sorted by similarity compared to the selected sequence on the selected region(s). Similarity is defined as the number of aminoacid acids at the same position divided by the number of aminoacids in selected regions. Clusters of identical regions appear on the same background.