There are three different ways to adjust the way Antibody Annotator and other pipelines identify the boundaries between FR and CDR regions. "Result Annotation Scheme" is the best option when using IMGT or Kabat, while the FR Adjustment option offers more fine control and is ideal for custom schemes. Using custom reference sequences is best if your CDR definitions differ significantly from IMGT definitions.
FR Adjustment is an advanced option and is is hidden by default. Please contact us if you would like it turned on.
Result Annotation Scheme
The first and simplest way is to choose between IMGT and Kabat definitions in the "Analysis options" section of Antibody Annotator.
This will automatically apply the CDR definitions for IMGT or Kabat as desired. Please contact us if you would like to see other schemes available in this option.
For more information on the regular annotator options, see here.
FR Adjustment option
The advanced FR Adjustment option allows you to customise your CDR definitions with fine control. You can set up CDR/FR boundary adjustments to create your own scheme. These adjustments are defined as amino acid offsets relative to the chosen numbering scheme, either IMGT or Kabat. Both positive and negative offsets are possible. This method would be most appropriate if you have small offsets.
This option is hidden by default, but is available for your organisation on request. Simply contact us if you would like it turned on.
There are two numerical input boxes for each FR region. Heavy chain FR regions and Light chain FR regions are addressed separately.
The first box for each region indicates the desired offset for the start of that FR region. "0" means no offset, or standard IMGT/Kabat region boundary. A negative offset will extend the FR region further to the left to include more amino acid(s), and a positive offset will make the FR region shorter by removing the first amino acid(s) from the FR region. So an offset of -1 in the very first box would add one amino acid to the start of the Heavy FR1 region.
The second box indicated the desired offset for the end of the FR region in a similar manner. "0" means no offset, a negative offset will shorten the FR region by one amino acid removing it from the end, and a positive offset will make the FR1 region longer by include more amino acid(s) at the end. In this case an offset of -1 would remove one amino acid from the end of the FR1 region. In this case, changing the end of the FR1 region will also affect the beginning of the CDR1 region. If you remove an amino acid from the end of the FR1 region, it will naturally get added to the start of the CDR1 region.
You might notice that a negative offset at the start adds an amino acid to the FR1 region, while a negative offset at the end removes an amino acid from the FR1 region. It might be easiest to think of the numbers as shifting the FR/CDR region boundary - negative moves the boundary to the left (5'end), positive moves it to the right.
Here is an example using the Sanger 1 tutorial dataset. The heavy chain initially looks like this with 0 offset (standard IMGT boundaries):
Next I will change the Region adjustments as below, by putting a -3 on the start of the Heavy FR4 region:
When I run this I get the following result:
You can see that the CDR3/FR4 boundary has moved 3 amino acids to the left. The three last amino acids of the CDR3 region MDV have now become the first three amino acids of the FR4 region.
Custom Reference Sequences
The final way to change your CDR definitions is to manually adjust them using custom reference germline sequences. By default, our pipelines assume that reference sequences are annotated using IMGT standard. However if you upload your sequences with alternate CDR/FR annotations then that scheme will be applied instead. To find out more about creating custom databases, see here.
Note: Using non-IMGT CDR/FR definitions in your reference sequences will break the "Result annotation scheme" option in Antibody Annotator. It is highly recommended to leave the "Result annotation scheme" option set to "IMGT" when using custom definitions.