How do I add additional annotations to my sequences?

The Antibody annotator has the capacity to add additional annotations such as HisTags, Signal peptides, Promoters and Terminators that are not part of the immunoglobulin scaffold.

To add these additional annotations, you can either choose to annotate these annotation from a Feature database or you can use the Find liabilities and assets function. Liabilities and Assets are great for identifying short, exact motifs in different sequence regions, whereas a Feature database is best if you have longer sequence features that may not match exactly.

Once you have selected your preferred choice of how to add these additional annotation, you can run the Antibody annotator analysis as per usual. The following outlines how the Feature database and Find liabilities and assets function can be used to annotate additional annotations.

Feature databases

To use this function, you will need to first create a Reference database containing annotated sequences with those additional annotations you would like to add. Note that only admin users can create Reference databases. Please refer to the following article to learn more on how to create reference sequence databases. When you do this, make sure to create a "Feature" type database.

Below is an example of a database containing a pIII and a PelB leader sequence involved in Phage display. You can add as many sequences as required into your database but note that we currently only support nucleotide sequences. You can also have reference sequences containing ambiguous bases but those sequences must have an unambiguous stretch of at least 10 nucleotides.

Once a Reference database with these additional features has been created, select the file that you wish to analyse, click Annotation and select Antibody annotator in the dropdown.

To annotate these additional features, select the Additional Features option in the Antibody annotator window and select your Reference database from the dropdown.

You can specify the percentage of mismatches between your query (input sequence) and the reference sequence by inputting the appropriate number in the Mismatches % field. In addition to this, you can also specify the Gap Size in the same manner.

Note that while the Feature database function is more suited for longer reference sequences it only supports nucleotide reference sequences. For protein reference sequences, please use the "Find Liabilities and Assets" field instead.

Find Liabilities and Assets

You can also add additional feature annotations to your sequences with the Find liabilities and assets function. To use this, you will need to input your additional feature sequence into the text box. We have a default set of additional feature which includes a 6XHis Tag, Myc Tag, M13 and PelB signal peptides.

Sequences will be annotated with the assets (in this case, the additional annotations) as per the motif specified in the text box. Below are some guidelines on how to add additional annotations:

• A motif starting with a full stop (.) indicates a nucleotide motif.
  
  • For example, 6His .CATCATCATCACCATCAC will find a nucleotide motif that is identical to the specified 6His motif anywhere in the sequence.
• A number in square brackets after a motif indicates the maximum number of mismatches allowed (gaps are not supported) when matching that motif.
  • For example, 6His .CATCATCATCACCATCAC [3] will find a nucleotide motif that is identical to the specified 6His motif or has 3 or less mismatches between the sequence and the specified 6His motif anywhere in the sequence
• Space separated region names in brackets after a motif list indicates that the motif is only applied if within those regions.
  • For example, 6His .CATCATCATCACCATCAC (FR1) will find the 6His nucleotide motif in the FR1 region only.
• If no region is specified, nucleotide motifs will be found anywhere within the sequence while amino acid motifs will only be found in frame in annotated regions. Use an asterisk (*) for the region for amino acid motifs to find them anywhere in any frame.
  • For example, 6His HHHHHH (*) will find a series of 6 consecutive histidines residues anywhere in the sequence in any translation frame.
• Prepend or append an annotation name with 3 full stops (...) to only find a motif before or after that annotation.
  • For example, 6His HHHHHH (FR4...) will find a series of 6 consecutive histidines residues downstream of FR4 while 6His HHHHHH (...FR4) will find a series of 6 consecutive histidines residues upstream of FR4.

Note that you can adjust the Asset score to any value that you prefer. Please refer to the following article to learn more about this Find liabilities and assets function and how to edit the options.

Please contact support if you would like any further details, we would be happy to help you find the best configuration.