![]() Comparing automated BASE analysis with manual analysis we confirmed the validity of BASE output: identity between manual and automated aBASE analysis was 100% for all outputs, except for immunoglobulin isotype determination. BASE consists of two modules: aBASE for immunological annotations and cloning primer lookup, and cBASE for plasmid sequence identity confirmation before expression. We developed BASE, an easy-to-use software for complete data analysis in single cell immunoglobulin cloning. Current protocols for generation of patient-derived recombinant monoclonal antibody libraries are time-consuming and contain repetitive steps, some of which can be assisted with the help of software automation. These are merely examples - you may add your own motifs of interest in this section.Repertoire analysis of patient-derived recombinant monoclonal antibodies is an important tool to study the role of B cells in autoimmune diseases of the human brain and beyond. Below, we’ve listed assets included by default in the Antibody Annotator tool options. Less likely amino acid pair in CDR2 region to undergo deamidation.Īntibody sequence assets are sequence motifs that are indicative of additions to the sequence that aid in cloning or downstream processing.Less likely amino acid pairs of Asparagine to trigger Asn to undergo deamidation.pH-dependent cleavage at the Threonine, Serine interface.The DP motif, as noted above in the -100 liabilities is associated with hydrolysis/cleavage. Deamidation of Asparagine to Aspartate, resulting in the motif DP.This is less prevalent than deamidation of the NG, NS and NA motifs. Deamidation of the Asparagine, Histidine motif.Asparagine linked glycosylation is associated with reduced conformational stability and reduced “shelf-life” of antibody products.Unpaired cysteines in specific antibody sequence regions may result in structural perturbation and introduce changes in hydrophobicity or apparent surface charges in proteins.Low_Quality_Base "Base Quality 0 (FR2 FR4 CDR1 CDR2 CDR3) Extra_Cysteine C>1 (FR1 FR3).High frequencies (>20%) of sequencing errors in critical VDJ and VJ antibody sequence regions may render annotation results unreliable. Sequencing errors are predicted by Phred (Q) scores reported in all fastq files.Possible_Sequencing_Error "Expected Sequencing Errors > 0.2" (VDJ-REGION VJ-REGION).Very high frequencies (>75%) of sequencing errors in critical VDJ and VJ antibody sequence regions will likely render annotation results unreliable. Likely_Sequencing_Error "Expected Sequencing Errors > 0.75" (VDJ-REGION VJ-REGION).This heterozygosity is indicative of low quality sequence i.e. Nucleotide heterozygosity or low quality reads at over 20 different base pairs in critical antibody regions.Contamination +R +S +Y +W +K +M +B +D +H +V "Base Quality >= 20" (VDJ-REGION VJ-REGION VDJC-REGION VJC-REGION).The Antibody annotator tool was unable to annotate this region of the sequence, due to poor sequence quality or incorrect reference database.Frame shift mutations are base pair insertions or deletions that change all subsequent downstream codons and result in non-functional proteins.These liabilities in the middle of an antibody sequence can result in an incomplete, non-functional antibody product. These are stop codons that terminate antibody translation.If you are interested in learning more about creating customised liabilities, you can read more here. Listed below are the liabilities included by default in the Antibody Annotator tool options. The Liabilities feature is found in the Antibody Annotator tool under Analysis Options, as shown below and can be turned on or off.Īntibody Annotator ranks the following liabilities on severity, from a score of +1 to -1000. How can I find liabilities in my sequences? These add a positive score of +1 and are discussed at the bottom of this article under the header Assets. *Note: Assets are also found when selecting the "Find liabilities and assets" analysis option in Antibody Annotator. Each liability adds a negative value to the overall score of a sequence* Liabilities will be shown as annotations on sequences and an overall "Score" will be given based on how many liabilities were found. Sequence motifs that may reduce the conformational stability or might otherwise impair the functioning of antibody products.Premature STOP codons or frameshift mutations.If you want to know how to specify your own custom liabilities, please see this article.Īntibody sequence liabilities and assets include: This article describes and outlines the default sequence liabilities found when running Antibody Annotator with the "Find liabilities and assets" analysis option turned on.
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