Antibody Sequence Liabilities

This article describes and outlines the default sequence liabilities found when running Biologics annotation pipelines with the Find liabilities and assets analysis option turned on. You can also Customize your Sequence Liabilities and Assets. If you are interested in how the positional liabilities based on antibody numbering work, please see our main article on this.

Jump to:

• What are liabilities?
• How can I find liabilities in my sequences?
• List of default liabilities
• Saving different sets of liabilities for different datasets
• Positional liabilities based on antibody numbering
• References
  
  

What are liabilities?

Antibody sequence liabilities and assets include:

• Likely sequencing errors (eg. poor quality sequences)
• Premature STOP codons or frameshift mutations
• Sequence motifs that may reduce the conformational stability or might otherwise impair the functioning of antibody products

Liabilities will be shown as annotations on sequences and an overall "Score" will be given based on how many liabilities were found. Each liability adds a negative value to the overall score of a sequence*

*Note: Assets are also found when selecting the "Find liabilities and assets" analysis option in Antibody Annotator. These add a positive score of +1 and are discussed at the bottom of this article under the header Assets.

How can I find liabilities in my sequences?

The Liabilities feature is found under Analysis Options when running Antibody Annotator, NGS Antibody Annotator or Single Cell Antibody Annotator and can be turned on or off.

The below default liabilities are ranked based on severity, from a score of +1 to -1000. The Score column of a Biologics Annotator Result document displays the summed score of the liabilities and assets specified. See Exploring the Columns of the All Sequences Table to learn more about what the output columns can tell you about your annotation analysis.

Listed below are the liabilities included by default in the Antibody Annotator tool options. If you are interested in learning more about creating customised liabilities, you can read more here.

Most Severe Liabilities (-1000)

• (Stop_Codon_(Amber) .TAG (In_Frame), Stop_Codon_(Ochre) .TAA (In_Frame), Stop_Codon_(Opal) .TGA (In_Frame))
  • These are stop codons that terminate antibody translation. These liabilities in the middle of an antibody sequence can result in an incomplete, non-functional antibody product. 
• Frame_Shift
  • Frame shift mutations are base pair insertions or deletions that change all subsequent downstream codons and result in non-functional proteins.
• Not_Fully_Annotated
  • The Antibody annotator tool was unable to annotate this region of the sequence, due to poor sequence quality or incorrect reference database.
• Contamination +R +S +Y +W +K +M +B +D +H +V "Base Quality >= 20" (VDJ-REGION VJ-REGION VDJC-REGION VJC-REGION)
  • Sequencing errors are predicted by Phred (Q) scores reported in all fastq files. High quality nucleotide heterozygosity where the Phred (Q) score is 20 or higher in critical antibody regions. This heterozygosity is indicative of sequence contamination. Each high quality heterozygous base will be flagged and a penalty of -1000 applied to the overall sequence score.
• Likely_Sequencing_Error "Expected Sequencing Errors > 0.75" (VDJ-REGION VJ-REGION)
  • Very high numbers (>0.75) of sequencing errors in critical VDJ and VJ antibody sequence regions will likely render annotation results unreliable. 
    • The error probabilities over the entire V(D)J region are summed, therefore there may be no specific base which is low quality
      
      

Severe Liabilities (-100)

• Possible_Sequencing_Error "Expected Sequencing Errors > 0.2" (VDJ-REGION VJ-REGION)
  • Sequencing errors are predicted by Phred (Q) scores reported in all fastq files. High numbers (>0.20) of sequencing errors in critical VDJ and VJ antibody sequence regions may render annotation results unreliable.
• Low_Quality_Base "Base Quality < 10" (VDJ-REGION VJ-REGION)
  • Low quality bases where the base quality Phred (Q) score is less than 10 in critical antibody regions. Each low quality base will be flagged with an annotation and a penalty of -100 applied to the overall sequence.
• Deamidation NG NS NA (Heavy_CDR2 Light_CDR1)
  
  • Deamidation of Asparagine in the following motifs: NG motif (Asparagine followed by Glycine), NS motif (Asparagine followed by Serine) or NA motif (Asparagine followed by Alanine). These motifs are associated with deamidation, a form of degradation, and can contribute to a shorter “shelf-life” of antibodies.
• Isomerization DG DS (Heavy_CDR2 Heavy_CDR3 Light_CDR1)
  
  • Isomerization of Aspartate in the following motifs: DG motif (Aspartate followed by Glycine), DS motif (Aspartate followed by Serine). These motifs are associated with Isomerization, a form of degradation, and can contribute to a shorter “shelf-life” of antibodies.
    
•  Cleavage DP
  • Cleavage at the Aspartate, Proline interface. This is a classic motif that can undergo pH-dependent hydrolysis.
    
• Oxidation M (CDR1 CDR2 CDR3)
  • Methionine oxidation in the CDRs. Oxidation in these specific sites is associated with reduced binding affinity and faster antibody product degradation. 
• Missing_Cysteine C<1 (FR1 FR3); Extra_Cysteine C>0 (FR2 FR4 CDR1 CDR2 CDR3); Extra_Cysteine C>1 (FR1 FR3)
  • Unpaired cysteines in specific antibody sequence regions may result in structural perturbation and introduce changes in hydrophobicity or apparent surface charges in proteins.
• Glycosylation N{P}S{P} N{P}T{P}
  • Asparagine linked glycosylation is associated with reduced conformational stability and reduced “shelf-life” of antibody products.
    
    

Medium Liabilities (-10)

• Deamidation NH
  • Deamidation of the Asparagine, Histidine motif. This is less prevalent than deamidation of the NG, NS and NA motifs.
• Hydrolysis NP
  • Deamidation of Asparagine to Aspartate, resulting in the motif DP. The DP motif, as noted above in the -100 liabilities is associated with hydrolysis/cleavage.
• Cleavage TS
  • pH-dependent cleavage at the Threonine, Serine interface.

• Hydrophobic_Pocket VVV WWW

  • Hydrophobic pocket anywhere on the sequence

• Hydrophilic_Pocket YY (Heavy_CDR3)
  

  • Hydrophilic pocket found to be undesirable in the HCDR3
    
    

Low liabilities (-1)

• Deamidation SN TN KN
  • Less likely amino acid pairs of Asparagine to trigger Asn to undergo deamidation. 
• Deamidation NY (Heavy_CDR2)
  • Less likely amino acid pair in CDR2 region to undergo deamidation.
    
    

+1 Assets

Antibody sequence assets are sequence motifs that are indicative of additions to the sequence that aid in cloning or downstream processing. Below, we’ve listed assets included by default in the Antibody Annotator tool options. These are merely examples - you may add your own motifs of interest in this section.

• 6His .CATCATCATCACCATCAC
  • Histidine tag for affinity purification
• Myc .GAACAAAAACTCATCTCAGAAGAGGATCTG
  • common epitope tag
• M13 .TTAGTTGTTCCTTTCTATTCTCACAGT
  • common sequencing primer
• Pel_B .TTACTCGCGGCCCAGCCGGCCATGGCC
  • signal peptide, indicates antibody localization
    

Saving different sets of liabilities for different datasets

Geneious Biologics allows you to save Profiles which can be used to record and re-run alternative settings depending on the dataset. This means that you can specify different sequence liabilities and other settings depending on what dataset you are working with.

Profiles can be saved and applied at the bottom of our Annotation analysis pipelines:

Positional Liabilities based on Antibody Numbering

The liabilities following the above +1 Assets have their own section, please see our main article: Positional Liabilities based on Antibody Numbering.

References