This article compares and contrasts how to analyze the same antibody sequence using Geneious Biologics and IgBLAST. This might be helpful for users that are unsure of how to find their way around a Biologics annotation result but are familiar with the IgBLAST output format. We also have a similar article for those who have used IMGT/V-Quest.
If you are new to Geneious Biologics, please see our main tutorials and Quick Start Guide for a more comprehensive introduction to using Geneious Biologics:
- Quick Start Guide
- Video Guide
- Workflows and Flowcharts for Antibody Analysis
- Sanger Tutorial 1. Assembling Chromatograms
- NGS Tutorial 1. Sequence Analysis
Jump to:
- A Brief Introduction to Geneious Biologics
- Input Sequence & Settings
- Identifying Germline Genes and "Score"
- Identifying Gene Identity% and Germline Mismatches
- Unlock deeper insights with Geneious Biologics
A Brief Introduction to Geneious Biologics
After logging in, you can see your folder tree on the left and your "workspace" on the right. At the top of your workspace you can see what folder you are in, along with various options like Export, Pre-processing, Annotation and Post-processing. Any documents (sequences, annotation results, alignments) contained in this folder will also be selectable here.
To upload your sequences in Biologics you can use the Upload Button (highlighted in orange below) or drag and drop files straight into a folder:
From there you can select Pre-processing options or go straight to Annotation. In this example, a single chain is used for simplicity. If you'd like to know more about our pre-processing options:
Input Sequence & Settings
A single chain is used for simplicity in this comparison. The sequence used is the VH chain of the antibody abituzumab, one of the sequences from the clinical antibodies dataset in Sanger Tutorial 2. Sanger Chain association.
The IgBLAST result was generated using the default settings available at NCBI. The Biologics result was generated using the default settings of Antibody Annotator, however Annotate germline gene differences and Find liabilities and assets was turned on under the Analysis Options:
Identifying Germline Genes and "Score"
IgBLAST Results
The following output is produced by IgBLAST:
According to IgBLAST, the closest matching germline genes are:
- IGHV1-46
- IGHD7-27
- IGHJ4
IgBLAST has also determined that the VDJ region is in-frame and that there are no stop codons.
Geneious Biologics Results
You can identify that the chain is fully annotated, in-frame and has no stop codons by looking at the Sequences Table. Biologics also Scores your antibody by identifying any liabilities that may be present in the sequence, such as sites of potential deamidation etc. You can find out more about the metrics used to score antibodies (and how to customize them) here: Antibody Sequence Liabilities.
There are a number of places you can find the closest germline gene matches in Biologics.
- Sequence Viewer
The sequences themselves will be annotated with the closest germline gene from your reference database in the Sequence Viewer. Make sure to select any sequences you want to view to bring them up in the Sequence Viewer.
Biologics has identified the same closest germline genes as IgBLAST, apart from the closest D gene which is identified as IGHD7-27 in IgBLAST and IGHD6 in Biologics. The D genes are often difficult to identify, as the corresponding region of the antibody undergoes significant rates of somatic hypermutation. - Sequences Table
In the All Sequences Table, the closest germline gene matches are recorded. You can search the table to find these columns. In the image below I've gone to Table Preferences on the right and searched "gene".
This has brought up all the columns which contain "gene" in the name.
You can jump straight to a column of interest quickly by hovering over a column in the list, and clicking the "focus column" box symbol that pops up.
The Sequences Table is highly customizable, click here to learn more. For example, you can:
- Toggle columns on and off using the checkboxes
- Re-arrange columns by dragging and dropping them
- Pin columns by right clicking on them in the All Sequences Table
- Save your preferences as "profiles" to quickly apply custom table settings in other documents
Identifying Gene Identity% and Germline Mismatches
IgBLAST Results
The table below from IgBLAST displays the number of DNA mismatches for different regions as compared to the closest-matching germline V gene.
IgBLAST also identifies the overall Identity% for the closest-matching germline genes as well as the DNA and amino acid mismatches:
Geneious Biologics Results
There are a number of places you can identify the Gene Identity% and the specific germline mismatches in Biologics.
- Sequence Viewer
By hovering over the gene annotations in the sequence viewer you can bring up the Identity% values for that gene:
The germline mismatches are also found as annotations on the sequence.
Note: On the image above only the amino acid variants are shown. You can toggle the DNA variant annotation track on and off in the Sequence Viewer Preferences.
- Sequences Table
You can easily recreate the IgBLAST table of mismatches in the CDR and FR regions in Biologics, while also tracking the number of amino acid mismatches.
Using the Table Preferences, I first turned off all columns, then searched CDR and turned on the following columns:
In the HCDR1, there are 5 DNA mismatches and 3 amino acid mismatches. I have also displayed the HGVS present.
Identity% in Biologics
You can also find the overall Identity% and the HGVS for each best-matched gene in the Sequences Table. I used Table Preferences to search for and select the columns below:
The Sequences Table is highly customizable, click here to learn more. For example, you can:
- Toggle columns on and off using the checkboxes
- Re-arrange columns by dragging and dropping them
- Pin columns by right clicking on them in the All Sequences Table
- Save your preferences as "profiles" to quickly apply custom table settings in other documents
Unlock deeper insights with Geneious Biologics
Biologics has many other capabilities not mentioned in this article. These are just a few of the other tools you can use for antibody discovery:
- You can align sequences to compare the amino acid diversity across a region or multiple regions: Sequence alignment
- Pairing chains is easy to do in Biologics and allows you to analyse the full V(D)J or V(D)JC regions together: Pairing Heavy and Light Chains
- Biologics has many tools to process NGS datasets, including Barcoded data and data with UMIs: Understanding Single Cell technologies: Barcodes and UMIs
- Our Annotation pipelines produce clusters by default. Clusters are used to group sequences together and aid in understanding large datasets. To learn more, see this article: What is a "cluster"?
- You can add your Assay Data (ELISA values etc.) to further inform your results: Adding Assay Data to your analysis results
- You can compare multiple different experiments to monitor clonal expansion or to compare similarities and differences in antibody repertoires between different subjects: Comparing Results across Multiple Experiments